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Purpose: Cutaneous melanoma is a common, aggressive cancer with increasing incidence. The identification of melanoma-specific deregulated genes could provide molecular markers for lymph node staging assays and further insight into melanoma tumorigenesis. Experimental Design:Total RNA isolated from 45 primary melanoma,18 benign skin nevi, and 7 normal skin tissue specimens were analyzed on an Affymetrix Hu133A microarray containing 22,000 probe sets. Results: Hierarchical clustering revealed a distinct separation of the melanoma samples from the benign and normal specimens. Novel genes associated with malignant melanoma were identified. Differential gene expression of two melanoma-specific genes, PLAB and L1CAM, were tested by a one-step quantitative reverse transcription-PCR assay on primary malignant melanoma, benign nevi, and normal skin samples, as well as on malignant melanoma lymph node metastasis and melanoma-free lymph nodes. The performance of the markers was compared with conventional melanoma markers such as tyrosinase, gp100, and MART1. Conclusion: Our study systematically identified novel melanoma-specific genes and showed the feasibility of using a combination of PLAB and L1CAM in a reverse transcription-PCR assay to differentiate clinically relevant samples containing benign or malignant melanocytes.
The design of functional molecules has been an active area of research in chemistry and molecular biology during the past decade, encompassing topics such as host-guest chemistry,1 ribozymes,2 catalytic antibodies,3 and most recently, fullerenes.4 Nucleolytic agents that cleave the phosphodiester backbone of DNA and RNA have been another important focus of activity.5 Interest in this subject has paralleled, and relied on, rapid advances in molecular biology, genome mapping and sequencing, and also the chemical synthesis of deoxyoligonucleotides and peptides. The goals of this research, the methodological approaches, and the insights into chemical biology provided by these molecules are the topic of this Account, which will emphasize research with the chemical nuclease activity of 1,10-phenanthroline-copper by our laboratory and others but will also summarize the innovative studies of Dervan and colleagues using ferrous-EDTA linked to targeting molecules.6-8Chemical nucleases are defined here as redox active coordination complexes that cleave DNA by an oxidative pathway. The term "nuclease" is used as a shorthand for "nucleolytic activity". Since these reagents are generally used in excess of their substrates and also self-destruct because they generate reactive intermediates, turnover has not been rigorously demonstrated. In addition to 1,10-phenanthroline-copper9 and ferrous-EDTA,10 other chelates capable of cleaving the phosphodiester backbone of DNA under physiological conditions include metalloporphyrins,11-13 uranyl acetate,14 and octahedral rhodium complexes.16-17 The generation of piperidine sensitive cleavage sites resulting from guanosine oxidation has been achieved with ruthenium18 and nickel complexes.19,20 Chemical methods for cleaving the phosphodiester backbone by hydrolysis or nucleophilic catalysis are not yet efficient enough to be useful.21-23 Nuclease Activity of 1,10-Phenanthroline-Copper. The nuclease activity of 1,10-phenanthrolinecopper (OP-Cu) was reviewed seven years ago in these David S. Sigman was born in New York, NY, In 1939. After receiving his Ph.D. at Harvard In 1965 with F. H. Westhelmer, he was a postdoctoral fellow with E. R. Blout at Harvard Medical School. Since joining the UCLA faculty In 1968, Ns research Interests have encompassed enzymatic reaction mechanisms, nucleic acid structures, DNA-protein interactions, and the design of site-specific nucleases. Thomas W. Bruice was bom In Los Angeles, CA, in 1952. After receiving his Ph.D. at UCSF In 1980 with D. V. Santl and G. L. Kenyon, he was a postdoctoral fellow with J. R. Knowles at Harvard. He became a resident in clinical pathology at UCLA after receiving an M.D. from the University of Miami in 1984. After postdoctoral work with D. S. Sigman, he joined Isis Pharmaceuticals. Abhijit Mazumder was born In Bombay, India, in 1965. He received his B.A. from Johns Hopkins University In 1986 and his Ph.D. at the University of Maryland in 1990 with J. A. Gerlt. He Is currently a postdoctoral fellow with D. S. Sigman at UCLA. Christ...
The DNA strand cleavage reaction catalyzed by endonuclease III from Escherichia coli (endo III) on the 3'-side of aldehyde abasic sites proceeds by a syn beta-elimination involving abstraction of the 2'-pro-S proton and formation of a trans alpha,beta-unsaturated aldose product; we previously reported the same stereochemical course for the reaction catalyzed by UV endonuclease V from bacteriophage T4 (UV endo V) [Mazumder, A., Gerlt, J. A., Rabow, L., Absalon, M. J., Stubbe, J., & Bolton, P. H. (1989) J. Am. Chem. Soc. 111, 8029-8030]. Since the UV endo V does not contain an 4Fe-4S center, the 4Fe-4S center present in endo III need not be assigned a unique role in the beta-elimination reaction. The beta-elimination reactions that occur under alkaline conditions (0.1 N NaOH) and in the presence of the tripeptide Lys-Trp-Lys proceed by anti beta-elimination mechanisms involving abstraction of the 2'-pro-R proton and formation of a trans alpha,beta-unsaturated aldose product. The different stereochemical outcomes of the enzymatic and nonenzymatic beta-elimination reactions support the hypothesis that the enzyme-catalyzed reactions may involve general-base-catalyzed abstraction of the 2'-pro-S proton by the internucleotidic phosphodiester leaving group.
The structures of a large number of HIV-1 integrase inhibitors have in common two aryl units separated by a central linker. Frequently at least one of these aryl moieties must contain 1,2-dihydroxy substituents in order to exhibit high inhibitory potency. The ability of o-dihydroxy-containing species to undergo in situ oxidation to reactive quinones presents a potential limitation to the utility of such compounds. The recent report of tetrameric 4-hydroxycoumarin-derived inhibitor 5 provided a lead example of an inhibitor which does not contain the catechol moiety. Compound 5 represents a large, highly complex yet symmetrical molecule. It was the purpose of the present study to determine the critical components of 5 and if possible to simplify its structure while maintaining potency. In the present study, dissection of tetrameric 5 (IC50 = 1.5 microM) into its constituent parts showed that the minimum active pharmacophore consisted of a coumarin dimer containing an aryl substituent on the central linker methylene. However, in the simplest case in which the central linker aryl unit consisted of a phenyl ring (compound 8, IC50 = 43 microM), a significant reduction in potency resulted by removing two of the original four coumarin units. Replacement of this central phenyl ring by more extended aromatic systems having higher lipophilicity improved potency, as did the addition of 7-hydroxy substituents to the coumarin rings. Combining these latter two modifications resulted in compounds such as 3,3'-(2-naphthalenomethylene)bis[4,7-dihydroxycoumarin] (34, IC50 = 4.2 microM) which exhibited nearly the full potency of the parent tetramer 5 yet were structurally much simpler.
Efficient replication of HIV-1 requires integration of a DNA copy of the viral genome into a chromosome of the host cell. Integration is catalyzed by the viral integrase, and we have previously reported that phenolic moieties in compounds such as flavones, caffeic acid phenethyl ester (CAPE, 2), and curcumin confer inhibitory activity against HIV-1 integrase. We now extend these findings by performing a comprehensive structure-activity relationship using CAPE analogues. Approximately 30 compounds have been prepared as HIV integrase inhibitors based on the structural lead provided by CAPE, which has previously been shown to exhibit an IC50 value of 7 microM in our integration assay. These analogues were designed to examine specific features of the parent CAPE structure which may be important for activity. Among the features examined for their effects on inhibitory potency were ring substitution, side chain length and composition, and phenyl ring conformational orientation. In an assay which measured the combined effect of two sequential steps, dinucleotide cleavage and strand transfer, several analogues have IC50 values for 3'-processing and strand transfer lower than those of CAPE. Inhibition of strand transfer was assayed using both blunt-ended and "precleaved" DNA substrates. Disintegration using an integrase mutant lacking the N-terminal zinc finger and C-terminal DNA-binding domains was also inhibited by these analogues, suggesting that the binding site for these compounds resides in the central catalytic core. Several CAPE analogues were also tested for selective activity against transformed cells. Taken together, these results suggest that the development of novel antiviral agents for the treatment of acquired immune deficiency syndrome can be based upon inhibition of HIV-1 integrase.
Seventeen lichen acids comprising despides, depsidones, and their synthetic derivatives have been examined for their inhibitory activity against HIV-1 integrase, and two pharmacophores associated with inhibition of this enzyme have been identified. A search of the NCI 3D database of approximately 200,000 structures yielded some 800 compounds which contain one or the other pharmacophore. Forty-two of these compounds were assayed for HIV-1 integrase inhibition, and of these, 27 had inhibitory IC50 values of less than 100 microM; 15 were below 50 microM. Several of these compounds were also examined for their activity against HIV-2 integrase and mammalian topoisomerase I.
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