Jeanne A. Burr

Epigenetic silencing of DSC3 is a common event in human breast cancer

IntroductionDesmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens.MethodsWe used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines.ResultsDSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure.ConclusionThese results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure.

ANTIOXIDANT STOICHIOMETRY AND THE OXIDATIVE FATE OF VITAMIN E IN PEROXYL RADICAL SCAVENGING REACTIONS

Oxidation ofR,R,R-α-tocopherol (vitamin E; TH) by peroxyl radicals generated from the azo initiator azobis(2,4-dimethylvaleronitrile) in acetonitrile, hexane, or in phospholipid liposomes yields 8a-(alkyldioxy)tocopherone adducts, 8a-(hydroxy)tocopherone, and their hydrolysis product α-tocopherolquinone TH oxidation also yields 4a,5-epoxy- and 7,8-epoxy-8a-(hydroperoxy)tocopherones and their respective hydrolysis products 2,3-epoxy-α-tocopherolquinone and 5,6-epoxy-α-tocopherolquinone. Previous work indicates that the distribution of TH oxidation products varies with reaction environment. We investigated the dependence of antioxidant stoichiometry on TH oxidation product distribution for reactions in hexane, acetonitrile, and in phosphatidylcholine liposomes. Yields of 8a-substituted tocopherones were highest in hexane and lowest in phosphatidylcholine liposomes. In contrast, yields of epoxide products were highest in the liposome system and lowest in hexane. Yields of α-tocopherolquinone were similar in all three systems. Antioxidant stoichiometry, measured by the inhibited autoxidation method, was approximately 2.0 peroxyl radicals trapped per TH consumed in acetonitrile and in liposomes. In hexane, a slightly larger stoichiometric factor of approximately 2.5 was measured. This may, in part, reflect the generation of more reactive alkoxyl radicals in hexane. The reaction environment thus markedly affects the balance between competing TH oxidation pathways but produces comparatively little effect on antioxidant stoichiometry. These results imply that competing reaction pathways contribute similarly to the antioxidant chemistry of TH.

Antioxidant reactions of α-tocopherolhydroquinone

Abstractα-Tocopherolhydroquinone (TQH2) is a product of α-tocopherol oxidation/reduction that exerts antioxidant effects in biological systems. TQH2 inhibited autoxidation of methyl linoleate initiated by peroxyl radicals derived from thermolysis of 2,2′-azobis(2,4-dimethylvaleronitrile) in acetonitrile. TQH2 oxidation yielded α-tocopherolquinone (TQ) as a major product and 2,3-epoxy-α-tocopherolquinone and 5,6-epoxy-α-tocopherolquinone as minor products. Each TQH2 consumed approximately two peroxyl radicals in the course of the oxidation. The data suggest that TQH2 scavenges peroxyl radicals primarily by electron transfer to form TQ and secondarily by addition-elimination to form the epoxyquinones.

Quantitation of vitamin K in human milk

A quantitative method was developed for the assay of vitamin K in human colostrum and milk. The procedure combines preparative and analytical chromatography on silica gel in a nitrogen atmosphere followed by reversed phase high performance liquid chromatography (HPLC). Two HPLC steps were used: gradient separation with ultraviolet (UV) detection followed by isocratic separation detected electrochemically. Due to co-migrating impurities, UV detection alone is insufficient for identification of vitamin K. Exogenous vitamin K was shown to equilibrate with endogenous vitamin K in the samples. A statistical method was incorporated to control for experimental variability. Vitamin K1 was analyzed in 16 pooled milk samples from 7 donors and in individual samples from 15 donors at 1 month post-partrum. Vitamin K1 was present at 2.94±1.94 and 3.15±2.87 ng/mL in pools and in individuals, respectively. Menaquinones, the bacterial form of the vitamin, were not detected. The significance of experimental variation to studies of vitamin K in individuals is discussed.

Antioxidant reactions of green tea catechins and soy isoflavones

Green tea, a popular beverage brewed from dried leaves of the tea bush (Camellia sinensis) is distinguished by the presence of a group of polyphenols called flavanols or catechins (Graham, 1992). In recent years, the principal green tea catechins, i.e. (-)-epicatechin, (-)-epicatechin-3-gallate, (-)-epigallocatechin (EGC), and (-)-epigallocatechin-3-gallate (EGCG) have been recognized to be effective protectants against certain forms of cancer (Katiyar et al., 1992; Yang et al., 1993). These protective effects often have been attributed to antioxidant actions (Jovanovic et al., 1994; Terao et al., 1994; Salah et al., 1995; Kondo et al., 1999). The potential role of soy products in cancer prevention also has received much attention in recent years. Epidemiological data indicate that consumption of soybean-containing diets is associated with a lower incidence of certain human cancers in Asian compared to Caucasian populations (King et al., 1980; Locke et al., 1980). Genistein is one of the two principal isoflavones found in soy (Murphy, 1982) and, like catechins, is thought to act in large part through its ability to scavenge oxidants involved in carcinogenesis (Wei et al., 1993).