Diets rich in fruits and vegetables are associated with lower risk of cancer which may be conferred in part by the antioxidant properties of these foods. However, antioxidant supplementation or increased consumption of antioxidant-rich foods has been reported to have inconsistent effects on DNA damage. The present work (the DART study) investigated the extent of inter-individual variation in DNA damage, the capacity for base excision repair (BER) and the responses of both variables to supplementation with an antioxidant supplement for 6 weeks. There was a wide inter-individual variation in endogenous lymphocyte DNA strand breaks (8-fold variation), in damage after a challenge with H2O2 (16-fold variation) and in DNA repair (41-fold variation) measured using the comet assay. When stratified into tertiles according to the pre-supplementation level of endogenous DNA damage, there was a statistically significant decrease in DNA damage after supplementation in the tertile with the highest pre-supplementation level of damage. There was no effect of supplementation on BER. Endogenous DNA damage level before supplementation was significantly different (P = 0·037) between the three genotypes for the Val16Ala single nucleotide polymorphism in manganese superoxide dismutase (rs4880) with individuals homozygous/wild type showing less damage than those carrying the alanine variant.

Selenium (Se), a dietary trace metal essential for human health, is incorporated into selenoproteins as selenocysteine. Selenoprotein P (SePP), the major plasma selenoprotein, has both transport and antioxidant functions. In humans, it exists in plasma as two isoforms of approximately 50 and 60 kDa. This study investigated the effect of polymorphisms in the SEPP-1 gene, Se supplementation, and disease status on the proportions of SePP plasma isoforms. SePP was isolated from plasma from healthy volunteers, before and after a 6-week supplementation with 100 microg sodium selenite, and from colon cancer patients and controls. SePP isoform distribution was analysed by Western blot. In healthy volunteers, the relative abundance of each isoform depended on two SEPP-1 polymorphisms: rs3877899, predicted to cause an Ala-to-Thr amino acid change at position 234, and rs7579, located in the 3'-untranslated region of SEPP-1 mRNA. The difference between genotypes disappeared after Se supplementation. A genotype-dependent reduction was seen in the proportion of the 60-kDa isoform in patients with colorectal cancer compared with controls. We conclude that functional polymorphisms in the SEPP-1 gene influence the proportion of SePP isoforms in plasma. An elevated proportion of the 60-kDa isoform of SePP may increase selenoprotein synthesis and reduce colorectal cancer risk.

Nucleotide excision repair (NER) is responsible for repairing bulky helix-distorting DNA lesions and is essential for the maintenance of genomic integrity. Severe hereditary impairment of NER leads to cancers such as those in xeroderma pigmentosum, and more moderate reductions in NER capacity have been associated with an increased cancer risk. Diet is a proven modifier of cancer risk but few studies have investigated the potential relationships between diet and NER. In the present study, the plasmid-based host cell reactivation assay was used to measure the NER capacity in peripheral blood mononuclear cells from fifty-seven volunteers aged 18–30 years before and after 6 weeks of supplementation with micronutrients (selenium and vitamins A, C and E). As a control, nine individuals remained unsupplemented over the same period. Volunteers were genotyped for the following polymorphisms in NER genes: ERCC5 Asp1104His (rs17655); XPC Lys939Gln (rs2228001); ERCC2 Lys751Gnl (rs13181); XPC PAT (an 83 bp poly A/T insertion–deletion polymorphism in the XPC gene). NER capacity varied 11-fold between individuals and was inversely associated with age and endogenous DNA strand breaks. For the first time, we observed an inverse association between adiposity and NER. No single polymorphism was associated with the NER capacity, although significant gene–gene interactions were observed between XPC Lys939Gln and ERCC5 Asp1104His and XPC Lys939Gln and ERCC2 Lys751Gnl. While there was no detectable effect of micronutrient supplementation on NER capacity, there was evidence that the effect of fruit intake on the NER capacity may be modulated by the ERCC2 Lys751Gnl single nucleotide polymorphism.

In eukaryotic cells, mRNA localization can provide local protein synthesis. Metallothionein-1 (MT-1) mRNA is associated with the perinuclear cytoskeleton, and this is essential for subsequent nuclear import of the protein. The present study defines the cis-acting localization signal and a trans-acting binding protein. Gel retardation and UV cross-linking assays using MT-1 3′UTR transcripts and CHO cell extracts revealed formation of a complex containing a ∼50-kDa protein. Only localization-positive mutant transcripts competed for binding of this protein. Using an RNA affinity technique, Western blotting, mass spectrometry, and a supershift assay, the protein was identified as Elongation factor 1α (eEF1α). Mutation and deletion analysis showed that two regions, nucleotides 21–36 and 66–76, were required for both binding and localization. RNA-folding prediction combined with chemical and enzymatic probing experiments suggest that these regions are in juxtaposition within a stem/internal loop structure. Mutations that are predicted to alter this structure abrogate protein binding. Our hypothesis is that the cis-acting signal in MT-1 3′UTR is formed by this stem/internal loop, that it binds eEF1α, and that eEF1α–cytoskeleton interactions play a role in perinuclear mRNA localization.

In eukaryotic cells, several mRNAs including those of c-myc and c-fos are localized to the perinuclear cytoplasm and associated with the cytoskeleton. The localization element of c-myc mRNA is present within its 3'UTR (3'-untranslated region) but the precise nature of this signal has remained unidentified. Chemical/enzymatic cleavage with RNases (ribonucleases) and lead have identified single-stranded and double-stranded regions in RNA transcripts of nucleotides 194-280 of the c-myc 3'UTR. Combined with computer predicted structure these results indicate that this region folds so that part of it forms a stem-loop structure. A mutation, that has been previously shown to prevent localization, leads to a different secondary RNA structure in this region as indicated by altered cleavage patterns. Competitive gel-retardation assays, using labelled transcripts corresponding to nucleotides 205-280 of c-myc 3'UTR, and fibroblast extracts revealed that the stem-loop region was sufficient for RNA-protein complex formation. In situ hybridization studies in cells transfected with reporter constructs, in which all or parts of the region corresponding to this stem-loop were linked to beta-globin, indicated that this region was sufficient for localization and that deletion of the nucleotides corresponding to the proposed upper-stem or terminal loop prevented localization. Our hypothesis is that an AU-rich stem-loop structure within nt 222-267 in the c-myc 3'UTR forms the perinuclear localization signal. Bioinformatic analysis suggests that this signal shares features with 3'UTRs of other localized mRNAs and that these features may represent a conserved form of signal in mRNA localization mechanisms.

Messenger RNA trafficking, which provides a mechanism for local protein synthesis, is dependent on cis-acting sequences in the 3' untranslated regions (3'UTRs) of the mRNAs concerned acting together with trans-acting proteins. The C-MYC transcription factor is a proto-oncogene product involved in cell proliferation, differentiation and apoptosis. Localization of c-myc mRNA to the perinuclear cytoplasm and its association with the cytoskeleton is determined by a signal in the 3'UTR. Here we show the specific binding of a trans-acting factor to the perinuclear localization element in the 3'UTR of c-myc mRNA and identify this protein as annexin A2. Gel retardation and UV cross-linking experiments showed that proteins in fibroblast extracts formed complexes with the region of c-myc 3'UTR implicated in localization; a protein of approximately 36 kDa exhibited specific, Ca(2+)-dependent binding. Binding was reduced by introduction of a mutation that abrogates localization. Using RNA-affinity columns followed by gel electrophoresis and mass spectrometry this protein was identified as annexin A2. The RNA-protein complex formed by cell extracts was further retarded by anti-(annexin A2). Purified annexin A2 bound to the same region of the c-myc 3'UTR but binding was reduced by introduction of a mutation, as with cell extracts. It is proposed that binding of annexin A2 to the localization signal in the c-myc mRNA leads to association with the cytoskeleton and perinuclear localization. The data indicate a novel functional role for the RNA-binding properties of annexin A2 in perinuclear localization of mRNA and the association with the cytoskeleton.

mRNA localization provides a mechanism for localized protein synthesis. mRNAs encoding certain proteins, including c-MYC, c-FOS, MT-1 (Metallothionein-1) and vimentin, are localized around the nuclei of mammalian cells and are associated with the cytoskeleton. Targeting of these mRNAs to the perinuclear cytoplasm is mediated by elements within their 3'-UTRs (3'-untranslated regions), but many of the trans-acting proteins remain unidentified. UV cross-linking assays using radiolabelled transcripts indicated that a protein of approx. 50 kDa (from the Chinese-hamster ovary cell extracts) bound to the MT-1 3'-UTR sequence. Competition experiments using unlabelled mutant 3'-UTR RNAs revealed that the binding of this protein is specific to localization-positive mutants. Isolation of a 50 kDa protein was achieved by an RNA affinity-based method in which biotinylated MT-1 3'-UTR RNA was anchored to paramagnetic beads. Bound proteins were eluted and analysed by SDS/PAGE. The 50 kDa protein was extracted from the gel, subjected to trypsin digestion and identified by matrix-assisted laser-desorption/ionization-time-of-flight mass spectrometry as eukaryote elongation factor 1alpha.

Subcellular localization of mRNAs is a key mechanism for the synthesis of proteins close to their site of function. The mRNA encoding MT-1 (metallothionein-1) is localized in the perinuclear cytoplasm, where it is associated with cytoskeletal-bound polysomes. This localization relies on sequences present in the 3′-UTR (3′-untranslated region). The present study aims to characterize the cis-acting localization element(s) within the 3′-UTR. Using transfected cells expressing tagged MT-1 differing in their 3′-UTRs (deleted or mutated), the section(s) of this region required for directing MT-1 transcripts to the perinuclear cytoplasm has been investigated. Different 3′-UTRs were also used in UV cross-linking experiments that highlighted two distinct regions (nt 26–30 and 66–76) necessary for the binding of a protein of approx. 50 kDa, presumably involved in the mRNA targeting. The poor sequence homology between the MT-1 3′-UTR of various species, together with the bipartite nature of the required cis-element, indicates the involvement of a particular structure in the localization signal. The secondary structure of the MT-1 3′-UTR was investigated using enzymic and chemical probing. Current structural analysis of mutant 3′-UTRs will allow the critical structural features of the MT-1 mRNA perinuclear localization signal to be defined.

Subcellular localization of mRNAs is a key mechanism for the synthesis of proteins close to their site of function. The mRNA encoding MT-1 (metallothionein-1) is localized in the perinuclear cytoplasm, where it is associated with cytoskeletal-bound polysomes. This localization relies on sequences present in the 3'-UTR (3'-untranslated region). The present study aims to characterize the cis-acting localization element(s) within the 3'-UTR. Using transfected cells expressing tagged MT-1 differing in their 3'-UTRs (deleted or mutated), the section(s) of this region required for directing MT-1 transcripts to the perinuclear cytoplasm has been investigated. Different 3'-UTRs were also used in UV cross-linking experiments that highlighted two distinct regions (nt 26-30 and 66-76) necessary for the binding of a protein of approx. 50 kDa, presumably involved in the mRNA targeting. The poor sequence homology between the MT-1 3'-UTR of various species, together with the bipartite nature of the required cis-element, indicates the involvement of a particular structure in the localization signal. The secondary structure of the MT-1 3'-UTR was investigated using enzymic and chemical probing. Current structural analysis of mutant 3'-UTRs will allow the critical structural features of the MT-1 mRNA perinuclear localization signal to be defined.