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Open Full Page [CANCER RESEARCH 61, 2307–2310, March 1, 2001] Distribution and Functional Consequences of Nucleotide Polymorphisms in the 3؅-Untranslated Region of the Human Sep15 Gene1 Ya Jun Hu, Konstantin V. Korotkov, Rajeshwari Mehta, Dolph L. Hatfield, Charles N. Rotimi, Amy Luke, T. Elaine Prewitt, Richard S. Cooper, Wendy Stock, Everett E. Vokes, M. Eileen Dolan, Vadim N. Gladyshev,2 and Alan M. Diamond2 Departments of Human Nutrition and Dietetics [Y. J. H., A. M. D.], Surgical Oncology [R. M.], and Hematology/Oncology [W. S.], University of Illinois at Chicago, Chicago, Illinois 60612; Department of Biochemistry, University of Nebraska, The Beadle Center, Lincoln, Nebraska 68588 [K. V. K., V. N. G.]; Section on the Molecular Biology of Selenium, Basic Research Laboratory, National Cancer Institute, NIH, Bethesda, Maryland 20892 [D. L. H.]; Department of Genetic Epidemiology, National Human Genome Center, Howard University, Washington DC 20059 [C. N. R.]; Department of Preventive Medicine and Epidemiology, Loyola University, Stritch School of Medicine, Maywood, Illinois 60153 [A. L., T. E. P., R. S. C.]; and Department of Medicine [E. E. V., M. E. D.], University of Chicago, Chicago, Illinois 60637 ABSTRACT translation requires a recognition element within the 3Ј-UTR3 (3) of the corresponding mRNA referred to as a SECIS element (7). SECIS ele- Selenium has been shown to prevent cancer in a variety of animal ments are present in the mRNAs of all of the selenocysteine-containing model systems. Both epidemiological studies and supplementation trials proteins and share structural features, including an approximately 80- have supported its efficacy in humans. However, the mechanism by which nucleotide stem-loop structure containing both an internal and apical loop selenium suppresses tumor development remains unknown. Selenium is of unpaired nucleotides and a conserved 4-bp domain within the stem. present in known human selenoproteins as the amino acid selenocysteine Although the functions of several selenoproteins have been determined, (Sec). Sec is inserted cotranslationally in response to UGA codons within to date there has not been definitive evidence associating any of these -selenoprotein mRNAs in a process requiring a sequence within the 3؅ untranslated region (UTR), referred to as a Sec insertion sequence (SE- proteins with either cancer risk or development. A recent search for novel selenium-containing proteins resulted in the isolation and characterization CIS) element. Recently, a human Mr 15,000 selenoprotein (Sep15) was identified that contains an in-frame UGA codon and a SECIS element in of a new selenoprotein (8). Examination of data contained in the dbEST the 3؅-UTR. Examination of the available cDNA sequences for this protein databases resulted in the assembly of a 1268 nucleotide cDNA with an revealed two polymorphisms located at position 811 (C/T) and at position ORF of 162 amino acids. Within this ORF was an in-frame UGA codon G/A) located within the 3؅-UTR. Here, we demonstrate significant corresponding to amino acid 93. This and the observation that this protein) 1125 75 differences in Sep15 allele frequencies by ethnicity and that the identity of labels with Se and contains a SECIS element in the 3Ј-UTR strongly the nucleotides at the polymorphic sites influences SECIS function in a support the conclusion that the Mr 15,000 selenoprotein (Sep15) contains selenium-dependent manner. This, together with genetic data indicating selenocysteine at position 93. Further analysis of the Sep15 cDNA loss of heterozygosity at the Sep15 locus in certain human tumor types, sequence indicated that it was well conserved through evolution; homol- suggests that Sep15 may be involved in cancer development, risk, or both. ogous genes exist in mouse, rat, Brugia malayi, Caenorhabditis elegans, and other animals. On the basis of the frequency of recovery of Sep15 cDNA sequences from particular libraries included in the dbEST data- INTRODUCTION base, it was concluded that Sep15 is expressed in a wide variety of human tissues, with the highest levels being observed in the thyroid and prostate Selenium is an essential trace element currently being considered as a (8, 9). Examination of the available cDNA sequences for this protein candidate chemopreventive agent. Research spanning the last 25 years revealed two distinct forms of the Sep15 gene differing by polymor- has established that selenium is effective in the reduction of cancer phisms located at position 811 (C/T) and at position 1125 (G/A) within incidence when provided to animals as doses only 5- to 10-fold above the the 3Ј-UTR (8). Here, we demonstrate significant differences in Sep15 nutritional requirement (1). Chemoprevention studies with selenium in allele frequencies by ethnicity and that the identity of the nucleotides at animal model systems have indicated that this element is effective for the polymorphic sites influences SECIS function in a selenium-dependent most, if not all of the organ systems and is protective against the manner. This, together with genetic data indicating LOH at the Sep15 carcinogenic effects of a wide variety of insults (1). Both epidemiological locus in certain human tumor types, suggests that Sep15 may be involved studies and supplementation trials have also supported its efficacy in in cancer etiology. humans (2–5). However, the mechanism by which selenium suppresses tumor development remains unknown. Many of the biological activities MATERIALS AND METHODS of selenium are attributable to its role as a constituent of selenium- containing proteins. In all of the known mammalian selenoproteins, Sample Sources. The majority of the samples obtained from African Amer- selenium exists as the amino acid selenocysteine, which is inserted icans, 490 subjects, was recruited from the community of Maywood, IL, a suburb of Chicago. Unrelated individuals over the age of 18 years were identified through cotranslationally into the nascent protein in response to the UGA codon door-to-door canvassing and asked to participate in a study of diet and heart in selenoprotein mRNA (6). In mammals, the molecular choice to insert disease from an IRB-approved protocol, and signed consent was given from all of selenocysteine into the elongating selenoprotein rather than terminate the individuals. Exclusion criteria included any known cancer, non-insulin-depen- dent diabetes mellitus, and heart disease. The lymphocytes from normal volunteers Received 8/30/00; accepted 12/28/00. and colon tumor samples were collected anonymously from an IRB-approved The costs of publication of this article were defrayed in part by the payment of page protocol. All subjects were Ͼ18 years of age. Normal Caucasian volunteers were charges. This article must therefore be hereby marked advertisement in accordance with recruited from the University of Chicago. Colon tumor samples were obtained 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by a grant from the American Institute for Cancer Research (to after surgical resection from patients with confirmed colon cancer. African- A. M. D.), a grant from the Cancer Research Foundation of America (to V. N. G.), and American patients with hematological malignancies were treated according to National Institute of Dental Research/National Cancer Institute Grant 11921 (to E. E. V.). institutional IRB-approved treatment studies for acute myeloid leukemia, acute 2 To whom requests for reprints should be addressed, Alan M. Diamond, Department of Human Nutrition and Dietetics, University of Illinois at Chicago, Chicago, IL 60612. Fax: (312) 413-0319; E-mail: [email protected]; or Vadim N. Gladyshev, Department of 3 The abbreviations used are: UTR, untranslated region; Sec, selenocysteine; SECIS, Biochemistry, University of Nebraska, The Beadle Center, Lincoln, NE 68588. Fax: selenocysteine insertion sequence; LOH, loss of heterozygosity; ORF, open reading (402) 472-4948; E-mail: [email protected]. frame; IRB, Institutional Review Board; EST, expressed sequence tag. 2307 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2001 American Association for Cancer Research. DISTRIBUTION AND FUNCTION OF Sep15 POLYMORPHISMS lymphoblastic leukemia, and chronic myeloid leukemia. All subjects were Ͼ18 for either SpeIorPstI(5Ј-AAAACTAGTGCTTTGTAACAGACTTGCG- years of age, were referred to the University of Illinois at Chicago for treatment, GTTAATTATGC-3Ј or 5Ј-AAAGTGCAGGGTCTTACAAATGATCACT- and consented to an IRB-approved study for the analysis of molecular abnormal- TTTAAATGGAC-3Ј, respectively). Amplification was as described above, ities present in leukemia cells. Bone marrow and blood samples were obtained at and the products were digested with SpeI and PstI and directionally cloned into specified time points before, during, and after completion of treatment. Breast the pBPLUGA plasmid. Transfection of mouse NIH 3T3 cells was performed carcinomas from African-American women were archived from the Department of using the lipofectin reagent at a ratio of 1:3 (v/v) as indicated by the vendor Surgical Oncology Tissue and Sera Bank at the University of Illinois at Chicago. (Life Technologies, Inc.). The cells were cotransfected with the reporter Originally, tumors were obtained from women with confirmed histopathological construct and the pSV2Neo plasmid at a 10:1 molar ratio, and transfectants diagnosis of breast cancer who were undergoing lumpectomy or mastectomy. were selected with the antibiotic geneticin G418. G418-resistant
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