The present study was designed to investigate the effects of two main constituents of green tea, (À)-epigallocatechin-3-gallate (EGCG) and caffeine, on intestinal tumorigenesis in Apc min/+ mice, a recognized mouse model for human intestinal cancer, and to elucidate possible mechanisms involved in the inhibitory action of the active constituent. We found that p.o. administration of EGCG at doses of 0.08% or 0.16% in drinking fluid significantly decreased small intestinal tumor formation by 37% or 47%, respectively, whereas caffeine at a dose of 0.044% in drinking fluid had no inhibitory activity against intestinal tumorigenesis. In another experiment, small intestinal tumorigenesis was inhibited in a dose-dependent manner by p.o. administration of EGCG in a dose range of 0.02% to 0.32%. P.o. administration of EGCG resulted in increased levels of E-cadherin and decreased levels of nuclear B-catenin, c-Myc, phospho-Akt, and phospho-extracellular signal-regulated kinase 1/2 (ERK1/ 2) in small intestinal tumors. Treatment of HT29 human colon cancer cells with EGCG (12.5 or 20 Mmol/L at different times) also increased protein levels of E-cadherin by 27% to 58%, induced the translocation of B-catenin from nucleus to cytoplasm and plasma membrane, and decreased c-Myc and cyclin D1 (20 Mmol/L EGCG for 24 hours). These results indicate that EGCG effectively inhibited intestinal tumorigenesis in Apc min/+ mice, possibly through the attenuation of the carcinogenic events, which include aberrant nuclear B-catenin and activated Akt and ERK signaling. (Cancer Res 2005; 65(22): 10623-31)

Gamma rays have been used to induce Chinese hamster ovary cell mutants in which the entire locus for dihydrofolate reductase (DHFR) has been eliminated. These mutants were isolated in two steps from a methotrexate-resistant clone (Flintoff, Davidson, and Siminovitch (1976). Somat. Cell Genet. 2, 245-262). The resistant cells contain amplified copies of a mutant dhfr gene that codes for a drug-resistant form of the enzyme. In the first step, methotrexate-sensitive mutants of the amplified line were selected. These mutants exhibit a reduced level of DHFR activity and contain a reduced number of dhfr genes. The remaining DHFR activity is methotrexate-sensitive. These mutants appear to be hemizygotes that have lost all copies of the amplified altered dhfr genes and retain one wild-type allele. In a second mutagenic step, mutants completely deficient in DHFR activity were isolated. Three of four of these mutants were the result of double deletions: they lack all traces of dhfr DNA sequences. The fourth mutant contains a deletion that extends through the 5' half of the dhfr gene. The hemizygotes for dhfr should be useful for the study of mutation at an autosomal mammalian locus without the complications of diploidy.

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.

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