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Nutrition and Disease

Both Selenoproteins and Low Molecular Weight Selenocompounds Reduce Colon Cancer Risk in Mice with Genetically Impaired Selenoprotein Expression¹

Robert Irons^*,, Bradley A. Carlson, Dolph L. Hatfield and Cindy D. Davis^*,2

^* Nutritional Science Research Group, National Cancer Institute, Rockville, MD 20852 and Molecular Biology of Selenium Section, Laboratory of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892

² To whom correspondence should be addressed. E-mail: davisci{at}mail.nih.gov.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Selenium has cancer protective effects in a variety of experimental systems. Currently, it is not known whether selenoproteins or low molecular weight selenocompounds are responsible for this activity. To evaluate the contribution of selenoproteins to the cancer protective effects of selenium, we used transgenic mice that carry a mutant selenocysteine transfer RNA gene, which causes reduced selenoprotein synthesis. Selenium homeostasis was characterized in liver and colon of wild-type and transgenic mice fed selenium-deficient diets supplemented with 0, 0.1, or 2.0 µg selenium (as selenite)/g diet. ⁷⁵Se-labeling, Western blot analysis, and enzymatic activities revealed that transgenic mice have reduced (P < 0.05) liver and colon glutathione peroxidase expression, but conserved thioredoxin reductase expression compared with wild-type mice, regardless of selenium status. Transgenic mice had more (P < 0.05) selenium in the nonprotein fraction of the liver and colon than wild-type mice, indicating a greater amount of low molecular weight selenocompounds. Compared with wild-type mice, transgenic mice had more (P < 0.05) azoxymethane-induced aberrant crypt formation (a preneoplastic lesion for colon cancer). Supplemental selenium decreased (P < 0.05) the number of aberrant crypts and aberrant crypt foci in both wild-type and transgenic mice. These results provide evidence that a lack of selenoprotein activity increases colon cancer susceptibility. Furthermore, low molecular weight selenocompounds reduced preneoplastic lesions independent of the selenoprotein genotype. These results are, to our knowledge, the first to provide evidence that both selenoproteins and low molecular weight selenocompounds are important for the cancer-protective effects of selenium.

KEY WORDS: • cancer • colon • selenium • selenoproteins • transgenic mice

The trace element selenium appears to have cancer-preventive properties based on a converging body of evidence from epidemiologic, clinical, and experimental studies (1,2). Supplemental selenium has been found to reduce the incidence and mortality of colon cancer in humans (3), the second most prevalent cause of death from cancer in the United States (4). This is consistent with animal studies showing the protective effects of selenium against aberrant crypt formation and colon tumor development (5–9). However, it is not currently known whether selenoproteins or low molecular weight selenocompounds mediate the protection. Although the cancer-protective effects of selenium in animals occur at a level of intake that is 10-fold greater than what is required to maximize the activity of numerous selenoproteins (10–12), many of the 24 selenoproteins identified in rodents, and 25 in humans (13), are still uncharacterized.

Selenoprotein transgenic mice, which have selectively reduced selenoprotein expression, may help determine the role of selenium and selenoproteins in cancer risk and prevention (14). Moustafa et al. (15) developed a transgenic mouse that carries a mutation at position 37 within the anticodon loop of selenocysteine transfer RNA (Sec tRNA^[Ser]Sec)³. Normally, position 37 contains an adenosine that is highly modified to N⁶-isopentenyladenosine (i⁶A). The i⁶A^–mutation inhibits the formation of the 2'-O-methyl group on the ribosyl moiety of 5-methoxycarbonylmethyluridine (mcm⁵U) at position 34, which is designated Um34 (16) and is the last step in the maturation process (17). The addition of Um34 is selenium dependent and thus is critical for the synthesis of those selenoproteins most responsive to selenium status [e.g., glutathione peroxidase 1 (GPX1)] (18). A reduction in selenoprotein synthesis occurs in a protein- and tissue-specific manner in i⁶A^– transgenic mice, whereby GPX1 and thioredoxin reductase 3 (TR3) are the most- and least-affected selenoproteins, respectively, and liver and testes are the most- and least-affected tissues, respectively, of the tissues examined (15). However, selenoprotein expression in the colon of these transgenic mice has not been determined. Furthermore, it is not known whether dietary selenium deficiency would exacerbate or selenium supplementation would abrogate the effects of the i⁶A^– mutant transgene.

To our knowledge, a selenoprotein knockout animal model has not been used to examine the effects of selenium on chemically induced carcinogenesis. Aberrant crypt foci are putative preneoplastic lesions that have been detected in human colon resections and in experimental animals treated with chemical carcinogens (19,20). Aberrant crypt foci are statistically associated with the number of tumors that ultimately develop (21–23). As such, they are excellent biomarkers for determining colon cancer risk without conducting a lengthy tumor study.

The purpose of these studies was to determine the role of selenoproteins and low molecular weight selenocompounds in preventing colon cancer risk. Selenium homeostasis was characterized as it relates to selenoprotein expression and activity in the colon of Sec tRNA^[Ser]Sec i⁶A^– transgenic mice. Furthermore, these studies assessed whether dietary selenium could compensate for altered selenoprotein metabolism in these transgenic mice. Carcinogen-induced aberrant crypt formation was used to determine the impact of altered selenoprotein expression in the presence or absence of supplemental dietary selenium.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Reagents. Unless stated otherwise, all reagents were purchased from Sigma. Azoxymethane was purchased from the NCI Chemical Carcinogen Reference Standard Repository; ⁷⁵Se (specific activity 1000 Ci/mmol) from the MU Research Reactor Facility (University of Missouri); [³H]serine (specific activity, 36 Ci/mmol) from Amersham; BCA (bicinchoninic acid) protein assay kit from Pierce; and DE-52 from Whatman. Aurothioglucose was obtained from Research Diagnostics; protein gels, PVDF membranes, and molecular weight markers from Invitrogen; and TCA (trichloroacetic acid) from Mallinckrodt Baker. Goat anti-actin, rabbit anti-GPX1, and rabbit anti-TR1 antibodies were purchased from Abcam; mouse anti-selenoprotein P from BD Pharminogen; donkey anti-rabbit IgG IRDye800 and donkey anti-mouse IgG IRDye 800 from Rockland; and donkey anti-goat IgG Alexa Flour 680 from Molecular Probes. Rabbit anti-GPX2 was a kind gift from Dr. Regina Brigelius-Flohe (German Institute of Human Nutrition), and rabbit anti-15 kDa selenoprotein (Sep15) was kindly provided by Vadim Gladyshev (Univerisity of Nebraska).

    Mice and diets. This study was approved by the Animal Care Committee of the NIH. Wild-type FVB/N and transgenic mice carrying a Sec tRNA^[Ser]Sec i⁶A^– transgene (designated herein as transgenic mice) in a FVB background were bred in accordance with the NIH institutional guidelines for the care and use of laboratory animals. Transgenic mice carry 40 copies of the mutant i⁶A^– transgene (15). Weanling mice were given deionized, autoclaved water and consumed ad libitum for 6 wk (experiments 1 and 2) or 15 wk (experiment 3). Mice were fed either a standard rodent diet, NIH-31A (NIH) (experiment 1), or a Se-deficient torula yeast basal diet (experiments 2 and 3) that was supplemented with sodium selenite to obtain 0 µg Se/g, 0.1 µg Se/g, or 2.0 µg Se/g diet (Harlan Teklad) as previously described (24). By analysis, the diets contained 0.002, 0.232, and 1.626 µg Se/g diet, respectively. The basal diet contained sucrose (538.84 µg/g diet), cellulose (50 µg/g diet), choline bitartrate (1 µg/g diet), vitamin A palmitate (0.016 µg/g diet), DL--tocopheryl acetate (0.09 µg/g diet), cholecalciferol (0.002 µg/g diet), and vitamin B-12 (0.04 µg/g diet).

In experiment 3, after 3 wk of being fed experimental diets, 11–15 mice/diet were given 4 weekly injections of azoxymethane (10 mg/kg body weight, subcutaneously). Mice ate the same diets for an additional 8 wk.

    Sample collection. Animals were killed by cervical dislocation and blood was collected by cardiac puncture. Livers were dissected into portions and stored at –70°C before analysis of radiolabeled selenoproteins, protein expression, enzymatic activity, tRNA species, and selenium concentrations. The colon was opened longitudinally and the mucosa was scraped off with a microscope slide. The muscle wall of the colon was used for selenium analysis. Samples were stored at –70°C prior to analysis. For aberrant crypt analysis, the entire large intestine was removed, opened, spread out with the lumen side up, cleaned, and fixed in 75% ethanol.

    Radiolabeled selenoproteins. Mice received intraperitoneal injections of 500 µCi ⁷⁵Se (as sodium selenite)/mouse, 24 h prior to tissue harvest. Liver and colonic mucosa were homogenized in 50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mL/L Igepal, and 25 mg/L protease inhibitor cocktail. Samples were sonicated, protein was determined by BCA reaction, and 20 µg of protein was separated by polyacrylamide gel electrophoresis on a 10% Bis-Tris gel, as described (25). The gel was dried and exposed to a PhosphorImager screen, which was developed using a Storm 840 PhosphoImager (Amersham).

    tRNA analysis. tRNA was isolated and analyzed as described previously (26). Colons from at least 10 mice were pooled to obtain enough sample for analysis. Briefly, total tRNA was isolated from tissues using phenol extraction and aminoacylated with [³H]serine under limiting tRNA conditions (26). The labeled seryl-tRNA was chromatographed twice, initially in the absence and then in the presence of Mg²⁺, on an RPC-5 column (27) as described (15,28). The [³H]serine content of each fraction was determined using a Packard model 2200CA liquid scintillation counter.

    Se analysis. Se concentrations in the liver, colon, and diet were determined by hydride-generation atomic absorption spectrometry according to a published procedure (29). Briefly, samples were prepared for analysis by predigestion in nitric acid and hydrogen peroxide, followed by high-temperature ashing in the presence of MgNO₃ as an aid to prevent Se volatilization. The protein precipitate and nonprotein fractions of liver were collected by extracting tissue homogenates with 0.25 volumes of TCA (trichloroacetic acid) containing 4 g/L sodium deoxycholate. The TCA precipitate contained primarily protein-bound selenium, whereas the homogenate contained low-molecular weight selenocompounds [see also reference (30)]. The Se concentrations in the protein fraction were determined by multiplying the analyzed Se concentration by the mass of the protein pellet divided by the mass of the tissue used for protein precipitation. Values are expressed as nmol Se/g of precipitated protein. Nonprotein fraction values were multiplied by the protein concentration and mass of the tissue used for extraction, and expressed as nmol Se/g liver.

    Enzyme activities. GPX activity was measured by the coupled assay procedure (31), which uses hydrogen peroxide as the substrate. This assay measures both GPX1 and GPX2 activity. Activity is expressed as units/mg protein, where 1 unit was defined as the amount of enzyme required to oxidize 1.0 µmol of NADPH · min^–1. TR activity was determined spectrophotometrically by the method of Holmgren and Bjornstedt (32) as modified by Hill et al. (33) and Hintze et al. (34). Activity was determined by subtracting the time-dependent increase in absorbance at 412 nm in the presence of the thioredoxin reductase activity inhibitor, aurothioglucose, from total activity. A unit of activity was defined as 1.0 µmol 5-thio-2-nitrobenzoic acid formed · min^–1. Protein concentrations were measured using the BCA reagent.

    Gel electrophoresis and Western blotting. Tissue extracts (25 µg protein/well) were loaded onto 10% Bis-Tris gels and separated by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes, which were blocked overnight at 4°C in 50 mL/L nonfat dry milk in Tris buffered saline with 1 mL/L Tween-20. Membranes were incubated for 2 h in 10 mL/L nonfat dry milk with primary antibodies (1 g/L): goat anti-actin or mouse anti-actin, and rabbit anti-GPX1, rabbit anti-GPX2, rabbit anti-TR1, or mouse anti-selenoprotein P. After 3 washes, membranes were incubated for 1 h with secondary antibodies (1 g/L): donkey anti-goat IgG Alexa Fluor 680 or goat anti-mouse IgG Alexa Fluor 680, and donkey anti-rabbit IgG IRDye 800, goat anti-rabbit IgG IRDye 800, or donkey anti-mouse IgG IRDye 800. After 6 washes, membranes were scanned using an Odyssey Imager and analyzed using Odyssey software v1.2 (both from LI-COR). Data are expressed as fluorescence intensity of the selenoprotein normalized to actin.

    Aberrant crypt formation. The fixed colon and rectum were stained with 1 g/L methylene blue in 0.1 mol/L sodium phosphate buffer (pH 7.4). Aberrant crypt foci and the total number of aberrant crypt were scored, without knowledge of the dietary treatment, by using a microscrope at a magnification of 10x to visualize the aberrant crypt foci (5).

    Statistical analysis. The data were analyzed by a 2-way ANOVA (diet selenium, genotype, and their interaction) using GraphPad Prism v2.0 (GraphPad Software). Bonferonni adjustments were used when comparing means to account for multiple comparisons when P < 0.05. The effect of genotype on selenoprotein activity was analyzed using an unpaired t test. Values are reported as means ± SEM. Means were considered significantly different at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Selenoprotein expression and activity. The main ⁷⁵Se-labeled proteins present in wild-type and i⁶A^– transgenic mice (Fig. 1) corresponded in molecular weight to TR1 and GPX1 (25). ⁷⁵Se labeling of TR1 was comparable in wild-type and transgenic mice. In contrast, ⁷⁵Se labeling of GPX1 was markedly increased in both liver and colon from wild-type compared with transgenic mice. In wild-type mice, ⁷⁵Se labeling of TR1 was greater in the colon than in the liver, while ⁷⁵Se labeling of GPX1 was greater in the liver than in the colon.

View larger version (80K): [in this window] [in a new window]   FIGURE 1  75Se-labeled proteins in liver and colon of wild-type (wt) and transgenic (i6A–) mice (n = 2/genotype) fed a standard rodent diet. Thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPX1) are indicated. Data are representative of 2 independent experiments.

    Dietary selenium and growth. A second study was conducted to determine whether selenium deficiency would exacerbate or supplemental selenium would compensate for altered selenoprotein metabolism in i⁶A^– transgenic mice. Mice were fed a basal torula yeast diet supplemented with 0, 0.1, or 2.0 µg Se (as selenite)/g diet (representing deficient, adequate, and supplemental selenium intake, respectively). Wild-type and transgenic mice had similar initial body weights and maintained similar weights regardless of selenium intake during the first 5 wk of the study. However, during the last week of the study, transgenic mice fed a selenium-deficient diet had a precipitous drop in body weight, resulting in a mean that was reduced (P < 0.05) at the termination of the study (23 ± 2 vs. 26 ± 1 g in the other groups). In this group, 5 of 6 mice exhibited signs of ataxia (i.e., hunched posture, clinched limbs, and inability to right themselves). These findings are consistent with previous observations (B. A. Carlson, V. N. Gladyshev, and D. L. Hatfield, unpublished observation). Two mice were euthanized 3 d prior to the end of the study because of the severity of their symptoms.

    Selenium analysis. The selenium concentrations of both liver and colon were higher (P < 0.001) when wild-type compared with i⁶A^– transgenic mice consumed adequate selenium (Table 2). In contrast, when wild-type compared with transgenic mice consumed supplemental selenium, liver selenium concentrations did not differ among the 2 genotypes. However, there was 50% less (P < 0.001) selenium in the colon of transgenic mice.

View larger version (9K): [in this window] [in a new window]   FIGURE 2  Percentage selenium in protein (A) and nonprotein fractions (B) of liver from wild-type and i6A– transgenic mice consuming diets containing deficient, adequate, or supplemental levels of selenium. Symbols represent individual values and the lines represent group means, n = 5–6 mice/diet · genotype–1. Means without a common letter differ, P < 0.05; ns, not significant.

View larger version (51K): [in this window] [in a new window]   FIGURE 3  Selenoprotein expression (A) and activity (B) in liver and colon of wild-type and i6A– transgenic mice consuming diets containing deficient, adequate, or supplemental levels of selenium. Representative Western blots of selenoproteins are shown above the actin blot, which was detected on the same membrane and serves as a loading control. Values represent means ± SEM, n = 5–6 mice/diet · genotype–1. Means without a common letter differ, P < 0.05.

    Azoxymethane-induced aberrant crypt formation and dietary selenium. The i⁶A^– transgenic mice weighed more (P < 0.0001) than wild-type mice at the start of the study (15.0 ± 0.7 vs. 10.3 ± 0.3 g, respectively) and at the end of the study (30.5 ± 0.8 vs. 25.5 ± 0.6 g, respectively). There was no significant effect of either dietary selenium or genotype on change in body weight during the study. The effect of dietary selenium on azoxymethane-induced aberrant crypt formation was determined in wild-type and transgenic mice (Fig. 4). Because of the weight loss exhibited by transgenic mice after 5 wk of a selenium-deficient diet, this group was not included in this experiment. Compared with wild-type mice on the same diet, transgenic mice had more (P < 0.05) aberrant crypts and aberrant crypt foci per colon. In both mouse strains, selenium supplementation decreased (P < 0.05) aberrant crypt and aberrant crypt foci formation. Indicators of selenium status in these mice showed that erythrocyte GPX activity was 0.7 ± 0.5, 4.7 ± 0.8, and 11.4 ± 1.3 units · mg protein^–1 for wild-type mice consuming selenium-deficient, adequate, and supplemental diet, respectively; and 0.7 ± 0.5 and 3.0 ± 1.7 units · mg protein^–1 for transgenic mice consuming selenium-adequate and supplemental diet, respectively. Only wild-type mice consuming supplemental selenium had increased (P < 0.001) erythrocyte GPX activity. Dietary selenium caused a dose-dependent increase in wild-type liver GPX activity (i.e., 0 ± 0.3, 122 ± 18, and 257 ± 15 units · mg protein^–1 for deficient, adequate, and supplemental selenium, respectively). Compared with wild-type mice on the same diet, GPX activity in the transgenic liver was decreased (P < 0.01), and did not signficantly increase in response to dietary selenium (3 ± 0.7 and 11 ± 2.6 units · mg protein^–1 for adequate or supplemental selenium, respectively). Compared with the liver, a greater percentage of selenium in colon was found associated with the low molecular weight, nonprotein fraction (ranging from 42 to 79%). Compared with the wild-type colon (P < 0.05; 54 ± 5%), more selenium was associated with the low molecular weight fraction of the transgenic colon (P < 0.05; 72% ± 4%).

View larger version (17K): [in this window] [in a new window]   FIGURE 4  Aberrant crypt (A) and aberrant crypt foci (B) per colon of azoxymethane-treated wild-type and i6A– transgenic mice consuming diets containing deficient, adequate, or supplemental levels of selenium. Values represent means ± SEM, n = 11–15 mice/diet · genotype–1. Means without a common letter differ, P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

GPX and TR are selenoproteins that control 2 major redox systems in the cell and have been implicated in cancer processes (35). Previous studies have shown that GPX1 and GPX2 double-knockout mice have increased chronic inflammation–induced colon cancer risk (36). About 25% of these mice naturally colonized with Helicobacter bacteria, when housed under nonspecific pathogen-free conditions, develop ileal and colonic tumors. Littermates that contained at least one wild-type GPX1 or GPX2 allele had no tumors (36). These and our results demonstrate that antioxidant activity of GPX is necessary to reduce cancer risk induced by chronic inflammation or colon carcinogens. However, we also observed a reduction in cancer risk in transgenic mice at a supranutritional level of selenium intake, indicating a beneficial effect of selenium that is independent of GPX expression. TR is thought to have opposing influences on inflammation-induced tumors, because the TR/thioredoxin system protects cells from oxidative damage, but also can act as an autocrine growth factor in tumor cells (37,38).

The liver and colon have opposite expression patterns of GPX1 and TR1, which may be explained by changes in methylation of the Sec tRNA^[Ser]Sec. Depending on selenium status, Sec tRNA^[Ser]Sec is variably methylated on the 2'-O-hydroxyl site of the ribosyl moiety at position 34, generating 2 isoforms: mcm⁵U and mcm⁵Um (39). Carlson et al. (18) show that Um34 of Sec tRNA^[Ser]Sec selectively regulates the expression of selenoproteins. In mice that express a mutant transgene, lacking i⁶A and Um34, GPX1 was not detected, whereas TR1 was expressed. Under conditions of selenium deficiency, there is a shift toward a predominance of the mcm⁵U isoform (40,41). Um34 synthesis requires the prior formation of modified bases: i⁶A, pseudouridine, 1-methyladenosine, and mcm⁵U (16). In wild-type mice, we confirmed that under conditions of adequate selenium, the Um34 isoacceptor is the predominate form in liver, but we found that mcm⁵U is the predominate form in the colon of wild-type mice. In contrast, in transgenic mice, Sec tRNA^[Ser]Sec in the liver is predominately mcm⁵U [see also reference (15)] and the percentage of mcm⁵U is increased in the colon. The limiting amount of Um34 in the colon of wild-type mice, and in the liver and colon of transgenic mice, contributes to the reduced GPX1 expression and conserved TR1 expression. It can be postulated that the relative contribution of selenoproteins and low molecular weight selenocompounds to cancer prevention will be different depending on the tissue and cellular environment and the form of selenium (inorganic vs. organic).

Previous studies suggest that low molecular weight selenocompounds may reduce cancer risk independent of selenoprotein activity. Whereas the activity of numerous selenoproteins reaches a maximum at adequate selenium intakes, selenoprotein activity does not change appreciably as dietary selenium is increased to the 10-fold higher level for cancer protective effects (10–12). A high intake of selenium increases the levels of methylated metabolites, including methylselenol, dimethyl selenide (expired in breath), trimethylselenonium (excreted in urine), and selenosugars (excreted in urine) (42). Ip and Ganther, et al. showed that methylated selenium metabolites that enter the selenium metabolic pathway downstream of hydrogen selenide, and do not provide selenium for selenoprotein synthesis, exhibit greater protection against cancer than compounds that could be converted to hydrogen selenide (43–45). Precursor selenium compounds (i.e., selenobetaine and Se-methylselenocysteine) that generate monomethylated selenium metabolites had the best cancer protective activity. In i⁶A^– transgenic mice, liver selenium was present at significantly higher amounts in the fraction not associated with protein. Compared with wild-type animals fed supplemental selenium, transgenic animals had twice the amount of selenium in the nonprotein fraction. This is most likely explained by the lack of GPX1 biosynthesis, which is the most abundantly expressed selenoprotein in liver and accounts for 60% of total hepatic selenium in selenium-adequate mice (46). This finding also implies that there is a greater amount of methylated selenium metabolites present in transgenic liver, which increases with greater dietary selenium intake. A greater amount of methylated selenium compounds also appeared to be present in transgenic colon, which could explain the decreased cancer risk we observed at supranutritional levels of selenium intake. However, it remains possible that the activity of some as yet unidentified selenoproteins may be playing a role or the two components may be acting in concert.

The relative impact of selenium in each form on cancer prevention may be tissue-specific and dictated by the Um34 distribution of the Sec tRNA^[Ser]Sec. Selenoproteins may be more important in tissues such as liver, which have a greater distribution of Um34, whereas low molecular weight selenocompounds may play a greater role in tissues like colon, which have a greater distribution of the unmethylated Sec tRNA^[Ser]Sec isoform (mcm⁵U). Future work is needed to determine the cancer-specific and tissue-specific roles of the 2 forms of selenium in mediating cancer protection. Use of tissue-specific selenoprotein (i.e., Sec tRNA^[Ser]Sec) conditional-knockout mice (28) will undoubtedly provide critical insight. Studies also need to determine whether dietary selenium can increase the Um34 content of tissues, as it does in liver (39). In conclusion, our studies implicate both selenoproteins and low molecular weight selenocompounds as playing important roles in the cancer protective effects of selenium in the colon. They also provide molecular evidence to support ongoing clinical trials that are testing whether supplemental selenium intake can decrease cancer risk in humans.

	ACKNOWLEDGMENTS

 
The authors express sincere thanks to Regina Brigelius-Flohé (German Institute of Human Nutrition, Potsdam-Rehbruecke, Germany) for providing the anti-GPX2 antibody, and to Vadim Gladyshev (University of Nebraska, Lincoln) for providing the anti-Sep15 antibody. We also thank John Finley (USDA, Grand Forks, ND) for the selenium analysis and John Milner (NCI, Rockville, MD) for his insight, discussion, and critical review of this article.

	FOOTNOTES

 
¹ This research was supported by the Division of Cancer Prevention and the Intramural Research Program of the NCI, NIH.

³ Abbreviations used: GPX, glutathione peroxidase; i⁶A^–, N⁶-isopentenyladenosine deficient; mcm⁵U, methylcarboxymethyl-5'-uridine; mcm⁵Um, methylcarboxymethyl-5'-uridine-2'-O-methylribose; Sec tRNA^[Ser]Sec, selenocysteine transfer RNA; Sep15, 15kDa selenoprotein; TR, thioredoxin reductase; Um34, methylcarboxymethyl-5'-uridine-2'-O-methylribose.

Manuscript received 13 December 2005. Initial review completed 17 January 2006. Revision accepted 13 February 2006.

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