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WO1999051637A1 - Selenoproteine mammalienne montrant une expression differentielle dans des cellules tumorales - Google Patents

Selenoproteine mammalienne montrant une expression differentielle dans des cellules tumorales Download PDF

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Publication number
WO1999051637A1
WO1999051637A1 PCT/US1999/007560 US9907560W WO9951637A1 WO 1999051637 A1 WO1999051637 A1 WO 1999051637A1 US 9907560 W US9907560 W US 9907560W WO 9951637 A1 WO9951637 A1 WO 9951637A1
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Prior art keywords
kda selenoprotein
kda
selenoprotein
nucleic acid
cdna
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PCT/US1999/007560
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English (en)
Inventor
Vadim N. Gladyshev
John C. Wootton
Alan Diamond
Dolph L. Hatfield
Kuan-Teh Jeang
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The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
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Application filed by The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services filed Critical The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
Priority to AU35497/99A priority Critical patent/AU3549799A/en
Publication of WO1999051637A1 publication Critical patent/WO1999051637A1/fr
Priority to US09/676,718 priority patent/US6849417B1/en
Priority to US10/919,554 priority patent/US7442543B2/en
Priority to US12/234,968 priority patent/US7776607B2/en
Priority to US12/814,933 priority patent/US20100255491A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to a mammalian selenocysteine-containing protein that is shown to be differentially expressed in tumor and non-tumor cells.
  • Methods of using the protein antibodies that bind to the protein and corresponding nucleic acid molecules are disclosed.
  • methods of using the nucleic acid molecules to determine the genotype of an individual is disclosed.
  • Selenium has been implicated in immunological function and many other biological processes through various nutritional and biochemical studies (Lee et al., 1997, Molecules & Cells 6:509-20: Hatfield et al., 1999, Comprehensive Natural Products Chemistry, 4, 353-80; Gladyshev and Hatfield, 1999, J. Biomed. Sci., in press). Recent studies have shown that supplementation of the diet with selenium resulted in 63% reduction in human prostate cancer and, to a lesser extent, in the reduction of colon and lung cancers (Clark et al., 1996, JAMA, 276: 1957-63). Selenium, a trace element, is a natural component of several prokaryotic and eukaryotic proteins.
  • selenium occurs in prokaryotic proteins either as a cofactor or as a selenocysteine residue
  • mammalian selenoproteins identified thus far contain selenium only in the form of selenocysteine, which is the 21st naturally occurring amino acid in protein.
  • a selenocysteine tRNA that decodes UGA has been found in all life kingdoms, suggesting that the use of UGA as a codon for selenocysteine is widespread in nature (Hatfield and Diamond, 1993, Trends Genet. 9:69-70).
  • Selenocysteine is located at the active center and is directly involved, or at least implicated, in the catalytic reactions catalyzed by glutathione peroxidases, thyroid hormone deiodinases and -2-
  • Thioredoxin reductase contains selenocysteine (Tamura and Stadtman, 1996, Proc. Natl. Acad. Sci. USA, 93: 1006-11) in a novel C-terminal Gly-Cys-Sec-Gly redox motif (Gladyshev et al., 1996, Proc. Natl. Acad. Sci. USA 93:6146-51). This center has been implicated in the peroxidase reaction catalyzed by the enzyme (Gladyshev et al., 1996, Proc. Natl. Acad. Sci.
  • the present invention relates to a newly isolated human protein of molecular weight around 15 kDa.
  • the protein contains a single selenocysteine residue, and is herein referred to as the 15 kDa selenoprotein.
  • the mouse homolog of the human 15 kDa selenoprotein is also provided.
  • the 15 kDa selenoprotein is shown to be expressed in a number of mammalian tissues, but is found at particularly high levels in prostate and thyroid tissues. Most notably, the expression of the 15 kDa selenoprotein and its mRNA are altered in several mammalian cancers.
  • the level of the protein was found to be 3-5 fold lower in tumorous hepatic cells in mice than in surrounding non- tumorous hepatic cells. Expression of the protein is also shown to be decreased in prostate cancer cell lines compared to healthy prostate cell lines.
  • the cDNA sequence of the human 15 kDa selenoprotein is 1244 nucleotides in length and contains an open reading frame encoding a 162 amino acid protein.
  • the 3' untranslated (UTR) region of the cDNA i.e., the region downstream of the ORF) contains a stem-loop selenocysteine insertion sequence (SECIS) element.
  • SECIS elements have been shown to be essential for insertion of selenocysteine into proteins at a UGA codon in coding sequences of other selenocysteine-containing proteins.
  • Two polymorphisms were detected in the 3' UTR of the human 15 kDa selenoprotein cDNA, one of which was located in the SECIS element.
  • a link between this polymorphism pattern and cancer was strongly suggested after the determination and subsequent analysis of the genotype of over 200 individuals.
  • a link was noted between the polymorphism and race.
  • One aspect of the invention is a purified preparation of the 15 kDa selenoprotein, as well as immunologically active fragments of this protein and specific binding agents, such as monoclonal antibodies, that specifically bind to the protein.
  • specific binding agents may be used to detect and quantify the presence of the 1 kDa selenoprotein in biological samples, and may be used in methods for detecting susceptibility to, or the presence of, cancer or monitoring the progression of the cancerous state.
  • a nucleic acid molecule encoding the 15 kDa selenoprotein, as well as probes and primers that are useful to detect and quantify the nucleic acid molecule.
  • Probes and primers that are useful to detect polymo ⁇ hisms in the cDNA sequence and the gene corresponding to the 15 kDa selenoprotein are also disclosed. Probes and primers that are useful to determine the genotype of an individual's 15 kDa selenoprotein are also disclosed. The detection of polymo ⁇ hisms in the 15 kDa selenoprotein cDNA or gene, and the determination of an individual's genotype, may be used to determine the susceptibility of an individual to cancer, including prostate cancer.
  • the invention also provides compositions and methods useful to determine the effect of chemical and biological agents (such as candidate tumor therapeutics) on the expression of the 15 kDa selenoprotein.
  • the effect of exposing cells to the candidate agent is assessed by measuring the change in expression levels of the 15 kDa selenoprotein mRNA or protein within the cell after exposure to the agent.
  • Such methods may be used to identify agents that have beneficial effects in the treatment or prevention of cancer, including prostate cancer.
  • FIG. 1 shows the human cDNA sequence encoding the 15 kDa selenoprotein and the amino acid sequence of the selenoprotein itself.
  • the putative signal peptide is shown in lower case and the most probable site of post-translational cleavage is indicated by an upward arrow.
  • the amino acid U represents selenocysteine 93 encoded by an in-frame TGA codon (overlined).
  • the sequences of four tryptic peptides, for which amino acid sequences were experimentally determined, are underlined.
  • the positions of the selenocysteine insertion sequence (SECIS element) and the poly-A addition signal (dotted underline) are shown.
  • FIG. 2 shows alignment of the human 15 kDa selenoprotein sequence with homologs from mouse, nematodes and rice.
  • FIGS. 3A and 3B relate to the SECIS element.
  • FIG. 3A shows the general features of eukaryotic SECIS elements used to identify a matching element in the 3'-UTRs of the mRNAs encoding human and mouse 15 kDa selenoproteins.
  • FIG. 3B shows an alignment of the predicted SECIS elements of the human and mouse mRNAs encoding the 15 kDa selenoprotein with a typical experimentally verified example (human GPX-1). In helical stems, single base bulges or mismatches are shown by gaps in the arrows. A lower case "a" residue above the human apical loop sequence indicates a polymo ⁇ hism at position 1125. -4-
  • FIG. 4 is a digital image of a Western blot showing the detection of the 15 kDa selenoprotein in cancerous and non-cancerous mouse liver tissues.
  • FIG. 5 is a digital image of a Western blot showing the detection of the 15 kDa selenoprotein in mouse cancerous and non-cancerous liver and prostate tissues.
  • FIG. 6 is a representative drawing showing the structure of the human 15 kDa selenoprotein cDNA. The C/T and G/A polymo ⁇ hisms at nucleotide positions 81 1 and 1125 respectively, are shown.
  • FIG. 7 is a digital image showing the use of primer extension (A) and restriction digestion
  • FIG. 8 A is a digital image showing the expression of recombinant forms of the 15 kDa selenoprotein, with Coomassie Blue staining showing the overexpression of the His-tag cysteine-for- selenocysteine mutant form of the 15 kDa selenoprotein.
  • FIG. 8B is a digital image showing expression of the His-tag selenocysteine-containing form of the 15 kDa selenoprotein.
  • Lanes 1-3 15 kDa selenoprotein cDNA; lanes 4-9: selenocysteine insertion sequence elements constructed downstream of TGA encoding selenocysteine (see FIG. 9 B and C).
  • Selenium-containing proteins were detected by metabolic labeling with 75 Se and visualized with a Phosphorlmager.
  • FIG. 9 shows the bacterial selenocysteine insertion sequence elements. These structures show the formate dehydrogenase H selenocysteine insertion sequence element (A) and two selenocysteine insertion sequence elements (B and C) designed downstream of the TGA codon encoding selenocysteine in the 15 kDa selenoprotein gene. The minimal essential structure necessary for selenocysteine inco ⁇ oration is boxed. 5'-end UGA encodes selenocysteine in these three constructs.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. In those sequence listings showing amino acid sequences, selenocysteine is represented by Xaa. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • Seq. I.D. No. 1 shows the amino acid sequence of the human 15kDa selenoprotein.
  • Seq. I.D. No. 2 shows the nucleic acid sequence of the human 15kDa selenoprotein cDNA.
  • Seq. I.D. No. 3 shows the nucleic acid sequence of the ORF of the human 15kDa selenoprotein cDNA.
  • Seq. I.D. No. 4 shows the amino acid sequence of the putative mature form of the human 15 kDa selenoprotein after post-translational modification.
  • Seq. I.D. Nos. 5-7 show examples of primers that may be used to amplify portions of the human 15 kDa selenoprotein cDNA.
  • Seq. I.D. No. 8 shows the nucleic acid sequence of the mouse 15 kDa selenoprotein cDNA.
  • Seq. I.D. No. 9 shows the amino acid sequence of the mouse 15 kDa selenoprotein.
  • Seq. I.D. Nos. 10 and 1 1 show examples of primers that may be used to amplify portions of the mouse 15 kDa selenoprotein cDNA.
  • Seq. I.D. Nos. 12 and 13 show examples of primers that may be used to amplify the polymo ⁇ hism containing region of human 15 kDa selenoprotein cDNA.
  • Seq. I.D. No. 14 shows a primer that can be used to determine the nucleotide at position 81 1 using primer extension.
  • Seq. I.D. No. 15 shows a primer that can be used to determine the nucleotide at position 1125 using primer extension.
  • cDNA complementary DNA: A piece of DNA lacking internal, non-coding segments
  • cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • 15 kDa selenoprotein A mammalian protein of approximate molecular weight 15 kDa that contains a selenocysteine residue encoded in the corresponding gene sequence by the codon UGA.
  • 15 kDa selenoprotein refers generically to mammalian 15 kDa selenoproteins; the specific human or murine forms are herein referred to as the "human 15 kDa selenoprotein” and the "murine” or "mouse 15 kDa selenoprotein.”
  • Mammalian 15 kDa selenoprotein polypeptides and cDNAs are orthologs of the disclosed murine and human 15 kDa sequences and are thus structurally related by the possession of similar amino acid and nucleic acid structures.
  • mammalian 15 kDa selenoprotein polypeptide sequences are characterized by possession of at least 70% amino acid sequence identity to the human 15 kDa selenoprotein amino acid sequence, determined using the BLAST program as described below.
  • Sequence identity the relatedness of two nucleic acid sequences, or two amino acid sequences is typically expressed in terms of the identity between the sequences (in the case of amino acid sequences, similarity is an alternative assessment). Sequence identity is frequently measured in terms of percentage identity; the higher the percentage, the more similar are the two sequences. Homologs of the human and mouse 15 kDa selenoproteins will possess a relatively high degree of sequence identity when aligned using standard methods.
  • NCBI Basic Local Alignment Search Tool (Altschul et al., 1990, J. Mol. Biol. 215:403-10) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at http://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
  • homologs When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are described at http://www.ncbi.nlm.nih.gov/BLAST blast_FAQs.html. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
  • the present invention provides not only the peptide homologs as described above, but also nucleic acid molecules that encode such homologs.
  • Stringent conditions are sequence dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5°C to 20°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al.
  • Nucleic acid molecules that hybridize under stringent conditions to a disclosed transcription factor cDNA sequence will typically hybridize to a probe based on either the entire cDNA or selected portions of the cDNA under wash conditions of 0.2x SSC, 0.1% SDS at 65°C.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequence that all encode substantially the same protein.
  • 15 kDa selenoprotein specific binding agent An agent that binds substantially only to a defined target.
  • a 15 kDa selenoprotein specific binding agent binds substantially only the 15 kDa selenoprotein.
  • 15 kDa selenoprotein specific binding agent includes anti-15 kDa selenoprotein antibodies and other agents that bind substantially only to the 15 kDa selenoprotein.
  • anti-15 kDa selenoprotein antibodies encompasses monoclonal and polyclonal antibodies that are specific for the 15 kDa selenoprotein, i.e., which bind substantially only to the 15 -8-
  • the anti-15 kDa selenoprotein antibodies used in the present invention are monoclonal antibodies (or immunologically effective portions thereof) and may also be humanized monoclonal antibodies (or immunologically effective portions thereof).
  • Immunologically effective portions of monoclonal antibodies include Fab, Fab', F(ab') 2 Fabc and Fv portions (for a review, see Better and Horowitz, 1989, Methods Enzymol. 178:476-96).
  • Anti-15 kDa selenoprotein antibodies may also be produced using standard procedures described in a number of texts, including "Antibodies, A Laboratory Manual” by Harlow and Lane, Cold Spring Harbor Laboratory (1988). The determination that a particular agent binds substantially only to the 15 kDa selenoprotein may readily be made by using or adapting routine procedures.
  • One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including "Antibodies, A Laboratory Manual” by Harlow and Lane. Cold Spring Harbor Laboratory, New York, 1988).
  • Western blotting may be used to determine that a given 15 kDa selenoprotein binding agent, such as an anti-15 kDa selenoprotein monoclonal antibody, binds substantially only to the 15 kDa selenoprotein, as described in Example 4, below.
  • a given 15 kDa selenoprotein binding agent such as an anti-15 kDa selenoprotein monoclonal antibody
  • Probes and primers Molecules useful as nucleic acid probes and primers may readily be prepared based on the nucleic acids provided by this invention. Typically, but not necessarily, such molecules are oligonucleotides, i.e., linear nucleic acid molecules of up to about 100 nucleotides in length. However, longer nucleic acid molecules, up to and including the full length of the 15 kDa selenoprotein cDNA may also be employed for such pu ⁇ oses.
  • a nucleic acid probe comprises at least one copy (and typically many copies) of an isolated nucleic acid molecule of known sequence that is used in a nucleic acid hybridization protocol. Generally (but not always) the nucleic acid molecule is attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various pu ⁇ oses are discussed, e.g., in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In: Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences, 1987).
  • Nucleic acid probes may be used in conjunction with array technologies to analyze the 15 kDa selenoprotein expression patterns in normal versus tumor cells.
  • a number of probes that are generally not conjugated to a detectable label or reporter molecule are affixed to a surface and hybridized with a sample nucleic acid preparation.
  • Primers are short nucleic acids, usually DNA oligonucleotides 8-10 nucleotides or more in length, and more typically 15-25 nucleotides in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase -9-
  • Primer pairs can be used for amplification of a nucleic acid sequence, e g , by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art
  • PCR polymerase chain reaction
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that pu ⁇ ose such as Primer (Version 0 5, ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, MA)
  • Primer Version 0 5, ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, MA
  • the invention thus includes isolated nucleic acid molecules that comprise specified lengths of the disclosed transcription factor cDNA sequences
  • Such molecules may comprise at least 8-10, 15, 20, 25, 30, 35, 40, 50, 75, or 100 consecutive nucleotides of these sequences and may be obtained from any region of the disclosed sequences
  • the human and mouse 15 kDa selenoprotein cDNAs shown in the Sequence Listing may be apportioned into halves or quarters based on sequence length, and the isolated nucleic acid molecules may be derived from the first or second halves of the molecules, or any of the four quarters
  • the human 15 kDa selenoprotein cDNA shown in Seq I D No 2 may be used to illustrate this This cDNA is 1244 nucleotides in length and so may be hypothetically divided into halves (nucleotides 1-622 and 623-1244) or quarters
  • Nucleic acid molecules may be selected that comprise at least 8-10, 15, 20, 25, 30, 35, 40, 50, 75 or 100 consecutive nucleotides of any of these portions of the transcription factor cDNA
  • one such nucleic acid molecule might comprise at least 25 consecutive nucleotides of the region comprising nucleotides 1-1244 of the disclosed transcription factor cDNA
  • a transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques
  • transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked D A by electroporation, hpofection, and particle gun acceleration
  • a vector may include nucleic acid sequences that permit it to replicate in the -10-
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • Isolated An "isolated" biological component (such as a nucleic acid or protein) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA and proteins.
  • Nucleic acids and proteins which have been "isolated” thus include nucleic acids and proteins purified by standard purification methods.
  • the term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Purified The term purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified 15 kDa selenoprotein preparation is one in which the 15 kDa selenoprotein is more enriched than the protein is in its natural environment within a cell.
  • a preparation of 15 kDa selenoprotein is purified such that the 15 kDa selenoprotein represents at least 50% of the total protein content of the preparation.
  • Oligonucleotide A linear polynucleotide sequence of up to about 100 nucleotide bases in length.
  • ORF open reading frame: A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Pharmaceutically acceptable carriers The pharmaceutically acceptable carriers useful in this invention are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • a recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • a therapeutically effective amount of the 15 kDa selenoprotein is defined as an amount that decreases a subject's risk of developing cancer, caused by the subject's increased predetermined genetic susceptibility to cancer associated with a polymo ⁇ hism in a 15 kDa selenoprotein gene.
  • Administration of a therapeutically effective amount of the 15 kDa selenoprotein will result in an increased amount of the 15 kDa selenoprotein in the subject, as compared to the amount of 15 kDa selenoprotein present prior to the administration of the 15 kDa selenoprotein.
  • Mammal This term includes both human and non-human mammals. Similarly, the terms “patient” and “subject” includes both human and veterinary subjects.
  • Amplify amplifying, amplification: Increasing the concentration of a nucleic acid in a sample.
  • One method will include the PCR reaction, which allows one to exponentially amplify the number of DNA molecules in a sample. Other methods may include, for example, dialysis. Other methods recognized by those skilled in the art are also included.
  • Tumors are abnormal growths which can be either malignant or benign, solid or liquid (for example, hematogenous). This term particularly includes malignant tumors which can be either solid (such as a breast, liver, or prostate carcinoma) or liquid (such as a leukemia). Tumors can also be further divided into subtypes, such as adenocarcinomas (e.g. of the breast, prostate or lung).
  • Cancer A malignant tumor.
  • Plasmids were isolated according to the instructions provided with the plasmid purification kit (Qiagen), the sequencing reaction products purified on separation columns as described by the manufacturer (Princeton Separations) and the nucleotide sequences of EST clones determined using a Dye Terminator Cycle Sequencing kit as described by the manufacturer (Perkin Elmer).
  • amino acids 145-162 was conjugated to the carrier KLH (keyhole limpet hemacyanin) and injected into rabbits. Specificity of the polyclonal antisera was determined using Western blotting of the purified recombinant human 15 kDa selenoprotein.
  • the human 15 kDa selenoprotein was detected in and purified from the human Jurkat T-cell line, JPX9 (Nagata et al, 1989, J. Virol. 63:3220-6) by growing the cells in the presence of 75 Se followed by analysis of extracts of the 75 Se-labeled cells by SDS PAGE and Phosphorlmager detection of radioactivity on the gels.
  • One of the major 75 Se-labeled proteins that migrated as a 15 kDa band on SDS PAGE was purified initially on DEAE-Sepharose and phenyl-Sepharose columns, and then further on a reverse-phase column. The procedures used were as follows.
  • JPX9 was grown and labeled with [ 7S Se]selenious acid (2 ⁇ Ci/ml) as described in Gladyshev et al. ( Proc. Natl. Acad. Sci. USA 93:6146-51. 1996).
  • 75 Se-labeled JPX9 cells were mixed with unlabeled cells, suspended in 2 volumes of 30 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 mM DTT, 1 mM MgCl 2 . 1 mM phenylmefhylsulfonyl fluoride and disrupted by sonication.
  • Disrupted cells were centrifuged, the supernatant applied to a DEAE-Sepharose column, which had been equilibrated with 30 mM Tris- HCl, pH 7.5, 2 mM DTT and 1 mM EDTA (buffer A), the column washed with 2 volumes of buffer -13-
  • Radioactive fractions corresponding to the 15 kDa selenoprotein eluted by application of a linear gradient from buffer A to water. Radioactive fractions were combined, concentrated, and loaded on a C I8 reverse-phase HPLC column that had been equilibrated in 0.05% trifluoroacetic acid (TFA), a gradient of 0 to 60% acetonitrile in 0.05% TFA applied and 75 Se-containing fractions corresponding to the 15 kDa selenoprotein eluted at 48% acetonitrile.
  • TFA trifluoroacetic acid
  • the molecular mass of the human 15 kDa selenoprotein subunit in fractions from the C 18 column determined by MALDI mass-spectrometry was 14,830 Da. Electrospray mass-spectrometry of the same preparation revealed a molecular mass of 14,870 Da. The N-terminus of the protein was blocked which prevented determination of the N-terminal sequence. Amino acid analysis of the purified protein (performed by Harvard Microchem, Boston,
  • MA shown in Table 1 , reveals a lack of internal methionine and histidine residues, as well as the hydrophobic character of the protein.
  • the 162 residue sequence corresponds to a full size open reading frame.
  • the 135 residue sequence corresponds to the open reading frame after removal of 27 N-terminal residues.
  • U represents selenocysteine.
  • sequences of three different tryptic peptides and one overlapping peptide from the 15 kDa selenoprotein were determined.
  • Computer searches of the partial cDNAs in the expressed sequence tags database (dbEST) using TBLASTN program revealed nucleotide sequences that corresponded to all three peptides in the same ORF. These cDNA sequences were used to assemble an open reading frame, depicted in FIG. 1.
  • the two cDNA clones containing the longest 5' sequences were obtained from I.M.A.G.E. Consortium at the Lawrence Livermore National Laboratory (California) (http://www-bio.llnl.gov/bb ⁇ /image/image.html) and sequenced.
  • N-terminal portion of the putative precursor of the 15 kDa selenoprotein as predicted from the gene sequence has a stretch of hydrophobic amino acid residues, suggesting the presence of a signal peptide Cleavage of these N-terminal amino acid residues is consistent with the amino acid composition of the protein (Table 1), since the processed protein matches more closely the ammo acid analysis data obtained for the purified 15 kDa selenoprotein than the full size 17 kDa protein
  • One possible site for post-translational processing is Ser27, which coincides with the site of an exon- lntron junction (not shown), making this residue the evolutionary favorable site for post-translational processing
  • the amino acid sequence of the mouse protein was deduced from the assembly of 39 independent partial cDNA sequences in dbEST
  • experimental confirmation of the 5' region encoding the mouse N-termmal sequence was made from partial cDNAs obtained from the IMAGE consortium
  • the C elegans sequence was assembled from two partial cDNA clones
  • mice and rat genes encode potential selenocysteine-containing 15 kDa proteins
  • the genes in C. elegans and B. malayi encode homologous proteins containing cysteine in place of selenocysteine.
  • nematode genes for glutathione peroxidase and thioredoxin reductase encode cysteine analogs of mammalian selenocysteine- containing proteins.
  • the complete mouse 15 kDa selenoprotein cDNA and amino acid sequences are presented in Seq. I.D. Nos. 8 and 9, respectively.
  • the 15 kDa selenoprotein gene exhibits a very broad spectrum of moderate expression in many tissues, and significantly higher levels of mRNA are shown by thyroid, parathyroid tumor, prostate and pre-cancerous prostate cells.
  • Expression estimates from dbEST library frequencies should be considered to be only semi-quantitative, considering that some libraries are normalized and variable levels of tissue contamination may exist. More quantitative representative estimates are given by the stringent CGAP (Cancer Gene Anatomy Project) libraries (Strausberg et al, 1997, Nature Genet. 15:415-6) prepared from small numbers of laser- microdissected cells, for example the pre-cancerous prostate library CGAP_Pr2 (Krizman et al,
  • Germinal B-cells (CGAP_GCB1) 1.0 (2/19194)
  • Helix I at least 4 base pairs
  • Internal loops 3-9 nucleotides
  • Pair the non- Watson-Crick base paired motif
  • UGAN following A in Internal loop
  • NGAN following the downstream strand of Helix II
  • Helix II 9-15 standard base pairs extending the quartet
  • Apical loop 10-20 nucleotides starting with AA(A/G). Single base mismatches or bulges were allowed within helices longer than 6 base pairs.
  • FIG. 3B shows these human and mouse sequences aligned with the canonical SECIS element (Low and Berry, 1996, Trends Biochem. Sci. 21 :203, and Walczak et al, 1996, RNA 2:367) of the human glutathione peroxidase 1 (GPX-1) mRNA 3'-UTR.
  • the 15 kDa protein mRNAs exhibit all the features known to be necessary in other eukaryotic selenoprotein mRNAs to promote selenocysteine insertion.
  • the expression of the 15 kDa selenoprotein and its mRNA is altered in several mouse and human cancers compared to non-cancerous tissues. Variations in the levels of both the polypeptide and the mRNA can be detected using standard procedures such as Western blotting (for polypeptide) and Northern blotting (mRNA).
  • the expression of the 15 kDa selenoprotein was compared in cancerous and non-cancerous mouse liver tissues by Western blotting using the polyclonal antibody described above.
  • equal amounts of protein were loaded on each lane in the following order: lanes 1 and 2, wild type, 2.5 months; lanes 3 and 4 - c-myc, 2.5 months; lanes 5 and 6 - c- myc/TGF ⁇ , 2.5 months; lanes 7 and 8- c-myc/TGF ⁇ , 10 months; lanes 9-1 1 - c-myc/TGF ⁇ , tumor, 10 months; lanes 12 and 13 - wild type, 1 month; lanes 14 and 15 - c-myc, 1 month; lanes 16 and 17 - c- myc/TGF ⁇ , 1 month; lanes 18 and 19- c-myc/TGF ⁇ , 10 months; lanes 20-22 - c-myc/TGF ⁇ , tumor.
  • c-myc/TGF ⁇ represents a double transgenic mouse.
  • the c-myc and c-myc/TGF ⁇ mice are models for accelerated hepatocarcinogenesis.
  • the levels of the 15 kDa selenoprotein polypeptide were observed to be 3-5 fold lower in tumor than in surrounding tissue in livers of c-myc/TGF ⁇ transgenic mice (FIG. 4).
  • These mice are characterized by elevated production of reactive oxygen species, increased lipid peroxidation and significant chromosome abnormalities.
  • Oxidative stress in c-myc/TGF ⁇ mice can be reduced by supplementation of the diet with vitamin E (V. Factor, personal communication), suggesting that selenium may have a similar protective effect.
  • expression of the 15 kDa protein was not altered in hepatocarcinomas of c-myc and c-myc/TGF ⁇ transgenic mice, for which no oxidative stress has been reported.
  • lane 1 - c-myc/TGF ⁇ liver 10 months (matched to the sample in lane 2); lane 2 - c-myc/TGF ⁇ liver, tumor 10 months; lane 3 - mouse prostate; lane 4 - purified human T-cell 15 kDa protein control 1 ; lane 5 - mouse prostate cancer cell line 1 ; lane 6 - mouse prostate cancer cell line 2; lane 7 - mouse prostate; lane 8 - c-myc/TGF ⁇ liver, 10 months (matched to the sample in lane 9); lane 9 - c-myc/TGF ⁇ liver, 10 months; lane 10 - purified human T-cell 15 kDa protein control 2.
  • Northern blotting revealed decreased expression of the human 15 kDa selenoprotein mRNA in matched samples from lymphoma and ovarian and fallopian tube cancers, and corresponding normal lymph node, ovary and fallopian tube (data not shown).
  • the genotype of the 15 kDa selenoprotein was determined for several individuals. Normal and cancerous tissues were collected, as well as blood samples to determine if the genotype of the tumor was different from that of non-tumor lymphocytes within the same individual. DNA from the blood and tissue samples was isolated using the protocols and procedures included in the Puragene DNA Isolation Kit (Gentra). The isolated DNA (0.1-1.0 ⁇ g) was used as template for Polymerase Chain Reaction (PCR) amplification using the GeneAmp PCR Amplification Kit and the following primers: forward primer 5'-CAGACTTGCGGTTAATTATG-3' (Seq. I.D. No. 12) and the reverse primer 5'-GCCAAGTATGTATCTGATCC-3' (Seq.
  • PCR Polymerase Chain Reaction
  • the PCR reactions included 0.2 mM dNTPs, 1.5 mM MgCl 2 , 0.4 mM each primer and 1.25 units of Taq polymerase and were incubated for 35 cycles of 85°C for 30 seconds, 45°C for 60 seconds, 72°C for 90 seconds).
  • radioactive primers were used.
  • the primer used was 5'-GGCATAGTAATCATCTGTCTTGTT-3' (Seq. I.D. No. 14), while the primer 5'-GTATGTATCTGATCCACACAAATCC-3' (Seq. I.D. No. 15) was used to determine the nucleotide at position 1125.
  • the primers were radiolabeled by 5'-end labeling with gamma-labeled ATP and polynucleotide kinase.
  • Labeled primer and DNA were mixed in a solution of 40 mM Tris HC1 (pH 7.5), 20 M MgCl 2 and 50 mM NaCl, heated to 95°C for 10 minutes, then transferred to a 37°C water bath for 1 hour. Extension was accomplished by the addition of 5 ⁇ l of a solution containing 100 mM DTT, 1 mM each of 3 dNTPs, 5 mM dideoxynucleotide triphosphate, and 5 units of reverse transcriptase or T7 polymerase. The mixture was further incubated for 15 minutes at 42°C, ethanol precipitated, resuspended in formamide loading buffer and the extension products separated on a 10% polyacrylamide gel. Visualization of the extension products was accomplished by autoradiography or phosphorimaging.
  • Figure 7 A shows the results of using primer extension with ddGTP to examine the polymo ⁇ hism at position 81 1.
  • the arrows show the primer extension products corresponding to two polymo ⁇ hic forms, C (lower arrow) or T (upper arrow) at position 81 1.
  • DNA from head and neck tumors was PCR amplified, isolated, and primer extended using Seq. I.D. No. 14.
  • the first lane contains primer only.
  • Lanes containing only the lower, shorter band are samples that are homozygous for C at position 811 (for example lanes 2-6).
  • Lanes containing only the upper, longer band are samples that are homozygous for T at position 81 1 (lane 21).
  • lanes containing both the lower and upper band are samples that are heterozygous (CT) at position 811 (for example lanes 7, 10, 12).
  • CT heterozygous
  • primer extension successfully allows for the determination of the genotype of the 15 kDa selenoprotein gene, the method is time consuming and requires the use of radioactive compounds. Therefore, an alternative method to determine the nucleotide identity at positions 81 1 and 1125 within the human 15 kDa selenoprotein gene was developed using restriction enzyme digestion.
  • the PCR amplified DNA generated above (0.5 ⁇ g) was digested with Dral (recognition sequence TTTAAA, Pharmacia), to evaluate the nucleotide identity at position 811 , or digested with Bfal (recognition sequence CTAG, New England Biolabs), to identify the nucleotide at position 1125, using buffers and conditions provided by the respective vendors. Evaluation of whether the D A was digested was accomplished by gel electrophoresis in 1 % agarose.
  • FIG. 7B shows DNA digested with Dral to identify the nucleotide at position 81 1. Only DNA containing a T at position 81 1 will be digested. Therefore, lanes containing only the upper band (lanes 2, 5, 6) are from individuals homozygous C at position 81 1 (compare to lane 8, undigested DNA). Lanes containing only the lower band (lane 4) are from individuals homozygous T at position 81 1 (both stands of DNA cut). Lanes containing both bands (lanes 1. 3 ,7) -21-
  • FIG. 7C shows DNA digested with Bfal to identify the nucleotide at position 1125. Only DNA containing a G at position 1125 will be digested. Lanes containing only the upper band are homozygous A (neither strand of DNA cut), lane containing only the lower band are homozygous G (both strands of DNA cut), while lanes containing both bands are heterozygous G/A at position 1 125.
  • CG/CG and TA/TA patients are homozygous at positions 81 1 and 1125 and CG/TA patients are heterozygous at positions 811 and 1125.
  • Table 3 the presence of the substitution polymo ⁇ hisms, T substituted for C at position 811 and A substituted for G at position 1125, were found more often in cancer samples, and is designated as a "cancer" polymo ⁇ hism.
  • the cancer polymo ⁇ hism therefore includes both the CG/TA and TA/TA alleles in Table 3.
  • the tendency of the cancer polymo ⁇ hism to be present in individuals having cancer was observed for the Caucasian population, and this observation was statistically significant for the African American population.
  • Table 3 also demonstrates that the cancer polymo ⁇ hism is more prevalent in the African American population.
  • an example of loss of heterozygosity has been detected in the sample of African American origin.
  • the African American population is known to be at higher risk of prostate cancer and dietary selenium (which may increase expression of the 15 kDa selenoprotein) has the single most pronounced effect in preventing this particular type of cancer.
  • the high expression of the 15 kDa protein in prostate tissue correlates with both the chemopreventive effect of selenium in the prostate, and the increased risk of prostate cancer in the African American population. Therefore, determination of an individual's genotype may be used as an indicator of the need for dietary selenium supplementation to inhibit tumor development.
  • this cancer polymo ⁇ hism may be used as the cancer predicting tool for populations at risk for developing certain cancers. -22-
  • the human 15 kDa selenoprotein was expressed in BL21(DE3) E. coli in the form of its cysteine-for-selenocysteine mutant (T for A substitution at nucleotide position 283), with (FIG. 8A) and without a His-tag using the pET-21b(+) vector (Novagen).
  • the human 15 kDa selenoprotein was genetically engineered to design a bacterial selenocysteine insertion sequence element (stem-loop structure downstream of the selenocysteine TGA codon), so that selenocysteine would be inco ⁇ orated into the human 15 kDa selenoprotein during its expression in bacteria.
  • the nucleotide sequence downstream of TGA encoding selenocysteine
  • RNA is extracted from human or mouse cells (e.g., hepatocytes) and used as a template for performing the reverse transcription-polymerase chain reaction (RT-PCR) amplification of cDNA.
  • RT-PCR reverse transcription-polymerase chain reaction
  • Primers may be chosen to amplify small segments of a cDNA or the entire cDNA molecule. Variations in amplification conditions may be required to accommodate primers of differing lengths, such considerations are well known in the art and are discussed in Innis et al. (PCR Protocols. A
  • the open reading frame of the human 15 kDa selenoprotein cDNA may be amplified using the following combination of primers: primer HI 5' ATGGCGGCTGGGCCGAGTGGG 3' (Seq. I.D. No. 5) and primer H2 5' TAATATGCGTTCCAACTTTTC 3' (Seq. I.D. No. 6), whereas that portion of the cDNA encoding the putative mature protein may be amplified using the following combination of primers: primer H3 5' TCTGCTTTTGGGGCAGAGTTT 3' (Seq. I.D. No. 7) and primer H2 5' TAATATGCGTTCCAACTTTTC 3' (Seq. I.D. No. 6).
  • the open reading frame of the mouse 15 kDa selenoprotein cDNA may be amplified using the following combination of primers: primer Ml 5'
  • primers are illustrative only; it will be appreciated by one skilled in the art that many- different primers may be derived from the provided cDNA sequences in order to amplify particular regions of the cDNAs.
  • the gene sequences corresponding to the cDNA sequences presented herein i.e. the genomic sequence including introns
  • pieces of such gene sequences may be obtained by amplification using primers based on the presented cDNA sequences using human or murine genomic DNA as a template.
  • PCR may also be used to produce variations on the 15 kDa selenoprotein cDN A sequences disclosed herein.
  • variants may be variants that are optimized for codon preference in a host cell that is to be used to express the protein, or other sequence changes that facilitate expression.
  • a sequence variant may be produced in which the TGA codon (encoding selenocysteine) is replaced with a codon encoding cysteine (either TCT or TGT).
  • TGA codon encoding selenocysteine
  • TGT codon encoding cysteine
  • cDNA sequence variant Two types may be produced.
  • the variation in the cDNA sequence is not manifested as a change in the amino acid sequence of the encoded polypeptide. These "silent" variations are simply a reflection of the degeneracy of the genetic code.
  • the cDNA sequence variation does result in a change in the amino acid sequence of the encoded protein, such as the U to C variation discussed above.
  • the variant cDNA sequence produces a variant polypeptide sequence.
  • any such amino acid substitutions are "conservative.” Conservative substitutions replace one amino acid with another amino acid that is similar in size, hydrophobicity, etc. Examples of conservative substitutions are shown in Table 4 below.
  • the immunologic identity of the protein may be assessed by determining whether it is recognized by an anti-15 kDa selenoprotein antibody: a variant that is recognized by such an antibody is immunologically conserved. Any cDNA sequence variant will -25-
  • genomic sequences may readily be obtained by standard laboratory methods, such as RACE-PCR amplification using a human genomic DNA library or genomic DNA extracted directly from human or murine cells as a template.
  • intron sequence data for the genomic sequence will be valuable for diagnostic applications, e.g., looking for splice-site mutations.
  • diagnostic applications e.g., looking for splice-site mutations.
  • the various applications described below e.g., expression of the 15 kDa selenoprotein for use in producing antibodies
  • 15 kDa selenoprotein cDNA sequence are described using a 15 kDa selenoprotein cDNA sequence, but may also be performed using the corresponding genomic sequence.
  • EXAMPLE 3 Expression and Purification of 15 kDa Selenoprotein Polypeptides
  • 15 kDa selenoprotein cDNA sequences With the provision of 15 kDa selenoprotein cDNA sequences, the expression and purification of corresponding 15 kDa selenoprotein polypeptides by standard laboratory techniques is now enabled.
  • the purified polypeptide may be used for functional analyses, antibody production and patient therapy.
  • the DNA sequence of the 15 kDa selenoprotein cDNA and the polymo ⁇ hic cDNAs disclosed above can be manipulated in studies to understand the expression of the gene and the function of its product. In this way, the underlying biochemical defect which results from mutation or reduced expression of the 15 kDa selenoprotein can be established.
  • polymo ⁇ hic versions of the 15 kDa selenoprotein cDNA isolated to date and others which may be isolated based upon information contained herein, may be studied in order to detect alteration in expression patterns in terms of relative quantities, tissue specificity and functional properties of the encoded 15 kDa selenoprotein.
  • this variant form of the protein is still referred to as the 15 kDa selenoprotein.
  • Methods for expressing large amounts of protein from a cloned gene introduced into Escherichia coli (E. coli) may be utilized for the purification, localization and functional analysis of proteins.
  • fusion proteins consisting of amino terminal peptides encoded by a portion of the E. coli lacZ or trpE gene linked to the 15 kDa selenoprotein may be used to prepare polyclonal and monoclonal antibodies -26-
  • these antibodies may be used to purify proteins by immunoaffinity chromatography, in diagnostic assays to quantitate the levels of protein, and to localize proteins in tissues and individual cells by lmmunofluorescence
  • the sequence variant or the native protein may also be produced in E coli in large amounts for functional studies
  • Methods and plasmid vectors for producing fusion proteins and intact native proteins in bacteria are described in Sambrook et al (In Molecular Cloning A Laboratory Manual, Cold Spring Harbor, New York, 1989, chapter 17)
  • Such fusion proteins may be made in large amounts, are easy to purify, and can be used to elicit antibody response
  • Native proteins can be produced in bacteria by placing a strong, regulated promoter and an efficient ⁇ bosome binding site upstream of the cloned gene If low levels of protein are produced, additional steps may be taken to increase protein production, if high levels of protein are produced, purification is relatively easy Suitable methods are presented in Sambrook et al (In Molecular Cloning A Laboratory Manual Cold Spring Harbor, New York, 1989) and are well known in the art Often, proteins expressed at high levels are found in insoluble inclusion bodies Methods for extracting proteins from these aggregates are described by Sambrook et al (In Molecular Cloning
  • Vector systems suitable for the expression of lacZ fusion genes include the pUR series of vectors (Ruther and Muller-Hill. 1983, EMBO J 2 1791), pEXl-3 (Stanley and Luzio, 1984, EMBO J 3 1429) and pMRlOO (Gray et al , 1982, Proc Natl Acad Sci USA 79 6598)
  • Vectors suitable for the production of intact native proteins include pKC30 (Shimatake and Rosenberg, 1981, Nature 292 128), pKK177-3 (Amann and Brosius, 1985, Gene 40 183) and pET-3 (Studiar and Moffatt, 1986, J Mol Biol 189 113) 15 kDa selenoprotein fusion proteins may be isolated from protein gels, lyophi zed, ground into a powder and used as an antigen
  • the DNA sequence can also be transferred to other cloning vehicles, such as other plasmids, bacte ⁇ ophages cos
  • the cDNA sequence need not be modified to remove the selenocysteine codon Rather, the 15 kDa selenoprotein cDNA may be directly hgated to heterologous promoters, such as the simian virus SV40 promoter in the pSV2 vector (Mulligan and Berg, 1981, Proc Natl Acad Sci USA 78 2072-6), and introduced into cells, such as monkey COS-1 cells (Gluzman, 1981, Cell 23 175-82), to achieve transient or long-term expression
  • the stable integration of the chime ⁇ c gene construct may be maintained in mammalian cells by biochemical selection, such as neomycin (Southern and Berg, 1982, J Mol Appl Genet 1 327-41 ) -27-
  • Normal mammalian cell growth medium contains sufficient trace selenium to permit efficient expression of the 15 kDa selenoprotein (for example, selenium is present in fetal bovine serum). However, the growth medium could be enriched if desired by the addition of selenite (Na 2 Se0 3 ).
  • DNA sequences can be manipulated with standard procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence- alteration via single-stranded bacteriophage intermediate or with the use of specific oligonucleotides in combination with PCR.
  • the cDNA sequence (or portions derived from it) or a mini gene (a cDNA with an intron and its own promoter) may be introduced into eukaryotic expression vectors by conventional techniques.
  • vectors are designed to permit transcription of the cD A eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription of the cDNA and ensure its proper splicing and polyadenylation.
  • Vectors containing the promoter and enhancer regions of the SV40 or long terminal repeat (LTR) of the Rous Sarcoma virus and polyadenylation and splicing signal from SV40 are readily available (Mulligan et al, 1981, Proc. Natl. Acad. Sci. USA 78:2072-6; Gorman et al, 1982, Proc. Natl. Acad. Sci USA 78:6777-81).
  • the level of expression of the cDNA can be manipulated with this type of vector, either by using promoters that have different activities, for example, the baculovirus pAC373 can express cDNAs at high levels in Spodptera frugiperda cells (Summers and Smith, 1985, In: Genetically Altered Viruses and the Environment, Fields et al. (Eds.) 22:319-28, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) or by using vectors that contain promoters amenable to modulation, for example, the glucocorticoid-responsive promoter from the mouse mammary tumor virus (Lee et al., 1982, Nature 294:228).
  • the expression of the cDNA can be monitored in the recipient cells 24 to 72 hours after introduction (transient expression).
  • some vectors contain selectable markers such as the g__ (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072-6) or neo (Southern and Berg, 1982, J. Mol. Appl. Genet. 1:327-41) bacterial genes. These selectable markers permit selection of transfected cells that exhibit stable, long-term expression of the vectors (and therefore the cDNA).
  • the vectors can be maintained in the cells as episomal, freely replicating entities by using regulatory elements of viruses such as papilloma (Sarver et al, 1981 , Mol. Cell Biol. 1 :486) or Epstein-Barr (Sugden et al, 1985, Mol. Cell Biol.
  • Th e transfer of DNA into eukaryotic, in particular human or other mammalian cells is now a conventional technique
  • the vectors are introduced into the recipient cells as pure DNA (transfection) by, for example, precipitation with calcium phosphate (Graham and vander Eb, 1973, Virology 52 466) or strontium phosphate (Brash et al , 1987, Mol Cell Biol 7 2013), electroporation (Neumann et al , 1982, EMBO J ⁇ 841), pofection (Feigner et al , 1987, Proc Natl Acad Sci USA 84 7413), DEAE dextran (McCuthan et al , 1968, J Natl Cancer lnst 41 351), micromjection (Mueller et al , 1978, Cell 15 579), protoplast fusion (Schafner, 1980, Proc Natl Acad Sci USA 77 2163-7), or pellet guns (Klein et al , 1987, Nature 327 70)
  • eukaryotic expression systems can be used for studies of the 15 kDa selenoprotein gene and variant forms of this gene, the 15 kDa selenoprotein and variant forms of this protein Such uses include, for example, the identification of regulatory elements located in the 5' region of the 15 kDa selenoprotein gene on genomic clones that can be isolated from human genomic DNA libraries using the information contained herein.
  • the eukaryotic expression systems may also be used to study the function of the normal complete protein, specific portions of the protein, or of naturally occurring or artificially produced mutant proteins
  • the expression vectors containing the 15 kDa selenoprotein gene or cDNA sequence or fragments or variants or mutants thereof can be introduced into human cells, mammalian cells from other species or non-mammalian cells as desired
  • human COS cells Gluzman, 1981, Ce// 23 175-82
  • monkey COS cells Gluzman, 1981, Ce// 23 175-82
  • Chinese hamster ovary CHO
  • mouse NIH 3T3 fibroblasts or human fibroblasts or lymphoblasts may be used
  • Expression of the 15 kDa selenoprotein in eukaryotic cells may be used as a source of proteins to raise antibodies
  • the 15 kDa selenoprotein may be extracted following release of the protein into the supernatant as described above, or, the cDNA sequence may be inco ⁇ orated into a eukaryotic expression vector and expressed as a chime ⁇ c protein with, for example, ⁇ -globm Antibody to ⁇ -globin is thereafter used to purify the chime ⁇ c protein
  • Corresponding protease cleavage sites engineered between the ⁇ -globin gene and the cDNA are then used to separate the two polypeptide fragments from one another after translation
  • One useful expression vector for generating ⁇ -globin chime ⁇ c proteins is pSG5 (Stratagene) This vector encodes rabbit ⁇ -globm
  • the present invention thus includes recombinant vectors comprising the selected DNA ot the DNA sequences of this invention (e g , the entire 15
  • control sequence may be selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof.
  • the expression control sequence may be specifically selected from the group consisting of the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the early and late promoters of SV40, promoters derived from polyoma, adenovirus, retrovirus, baculovirus and simian virus, the promoter for 3 -phosphogly cerate kinase, the promoters of yeast acid phosphatase, the promoter of the yeast alpha-mating factors and combinations thereof.
  • the host cell which may be transformed with the vector of this invention, may be selected from the group consisting of bacteria; yeast; fungi; plant; insect; mouse or other animal; or human tissue cells.
  • mutant or variant DNA sequences similar systems are employed to express and produce the mutant or variant product
  • Monoclonal or polyclonal antibodies may be produced to the 15 kDa selenoprotein or portions thereof.
  • antibodies raised against the 15 kDa selenoprotein will specifically detect the 15 kDa selenoprotein. That is, antibodies raised against the 15 kDa selenoprotein would recognize and bind the 15 kDa selenoprotein and would not substantially recognize or bind to other proteins found in human cells.
  • the determination that an antibody specifically detects the 15 kDa selenoprotein is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al. In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).
  • a given antibody preparation such as one produced in a mouse against the human 15 kDa selenoprotein specifically detects the 15 kDa selenoprotein by Western blotting
  • total cellular protein is extracted from human cells (for example, lymphocytes) and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel.
  • the proteins are then transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane.
  • the presence of specifically bound antibodies is detected by the use of an anti-mouse antibody conjugated to an enzyme such as alkaline phosphatase; application of the substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immuno-localized alkaline phosphatase.
  • Antibodies which specifically detect the 15 kDa selenoprotein will, by this technique, be shown to bind substantially only the 15 kDa selenoprotein band (which will be localized at a given position on the gel determined by its molecular weight). Non-specific binding of the antibody to other proteins may occur and may be -30-
  • Antibodies that specifically bind to the 15 kDa selenoprotein belong to a class of molecules that are referred to herein as "specific binding agents.”
  • Specific binding agents that are capable of specifically binding to the 15 kDa selenoprotein may include polyclonal antibodies, monoclonal antibodies (including humanized monoclonal antibodies) and fragments of monoclonal antibodies such as Fab, F(ab')2 and Fv fragments, as well as any other agent capable of specifically binding to the 15 kDa selenoprotein.
  • Substantially pure 15 kDa selenoprotein suitable for use as an immunogen is isolated from transfected or transformed cells as described above.
  • Concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms per milliliter.
  • peptide fragments of the 15kDa selenoprotein may be utilized as immunogens. Such fragments may be chemically synthesized using standard methods, or may be obtained by cleavage of the whole 15kDa selenoprotein followed by purification of the desired peptide fragments.
  • Peptides as short as 3 or 4 amino acids in length are immunogenic when presented to the immune system in the context of a Major Histocompatibility Complex (MHC) molecule, such as MHC class I or MHC class II.
  • MHC Major Histocompatibility Complex
  • peptides comprising at least 3 and pereferably at least 4, 5, 6 or more consecutive amino acids of the disclosed 15 kDa selenoprotein amino acid sequences may be employed as immuogens to raise antibodies.
  • naturally occurring epitopes on proteins are frequently comprised of amino acid residues that are not adjacently arranged in the peptide when the peptide sequence is viewed as a linear molecule, it may be advantageous to utilize longer peptide fragments from the 15 kDa selenoprotein amino acid sequences in order to raise antibodies.
  • peptides that comprise at least 10, 15, 20. 25 or 30 consecutive amino acid residues of the 15 kDa selenoprotein amino acid sequence may be employed.
  • Monoclonal or polyclonal antibodies to the intact 15 kDa selenoprotein or peptide fragments of this protein may be prepared as described below.
  • Monoclonal Antibody Production by Hybridoma Fusion Monoclonal antibody to epitopes of the 15 kDa selenoprotein identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495, 1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth -31-
  • Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Enzymol. 70:419, 1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). In addition, protocols for producing humanized forms of monoclonal antibodies (for therapeutic applications) and fragments of monoclonal antibodies are known in the art.
  • Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein, which can be unmodified or modified to enhance immunogenicity.
  • Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant.
  • host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intraderma! sites appear to be most reliable.
  • An effective immunization protocol for rabbits can be found in Vaitukaitis et al. (J. Clin.
  • Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony et al. In: Handbook of Experimental Immunology, Wier, D. (ed.) chapter 19, Blackwell, 1973. Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 ⁇ M). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (1980).
  • Antibodies Raised by Injection of 15 kDa Selenoprotein cDNA Antibodies may be raised against the 15 kDa selenoprotein by subcutaneous injection of a
  • DNA vector which expresses the 15 kDa selenoprotein into laboratory animals, such as mice. Delivery of the recombinant vector into the animals may be achieved using a hand-held form of the Biolistic system (Sanford et al, 1987, Particulate Sci. Technol. 5:27-37) as described by Tang et al. (Nature 356: 152-4, 1992). Expression vectors suitable for this pu ⁇ ose may include those which express the 15 kDa selenoprotein cDNA under the transcriptional control of either the human ⁇ -actin promoter or the cytomegalovirus (CMV) promoter.
  • Methods of administering naked DNA to animals in a manner to cause expression of that DNA in the body of the animal are well known and are described, for example, in U.S. Patent Nos. 5,620.896 ("DNA vaccines against rotavirus -32-
  • Antibody fragments may be used in place of whole antibodies and may be readily expressed in prokaryotic host cells. Methods of making and using immunologically effective portions of monoclonal antibodies, also referred to as antibody fragments, are well known and include those described in Better and Horowitz (Methods Enzymol. 178:476-496, 1989). Better et al. (Advances in Gene Technology: The Molecular Biology of Immune Disease & the Immune Response (ICSU SHort Reports), Streilein et al, eds. vol. 10: 105. 1990). Glockshuber et al. (Biochemistry 29: 1362-7, 1990), and U.S. Patent Nos.
  • Humanized monoclonal antibodies are preferred in clinical applications. Methods of making humanized monoclonal antibodies are well known, and include those described in U.S. Patent Nos. 5,585,089 ("Humanized Immunoglobulins"), 5,565,332 ("Production of Chimeric Antibodies ⁇ A Combinatorial Approach"), 5,225,539 ("Recombinant Altered Antibodies And Methods Of Making Altered Antibodies”), 5,693,761 ("Polynucleotides Encoding Improved Humanized Immunoglobulins"), 5,693,762 (“Humanized Immunoglobulins”), 5.585,089 (“Humanized Immunoglobulins”), and 5,530.101 ("Humanized Immunoglobulins”), and references cited therein.
  • Antibody preparations prepared according to these protocols are useful in quantitative immunoassays to determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.
  • 15 kDa selenoprotein cD A sequence information presented herein is in the area of genetic testing, carrier detection and prenatal diagnosis for mutations in the 15 kDa selenoprotein gene sequence.
  • Individuals carrying mutations in the 15 kDa selenoprotein gene may be detected at the DNA or RNA level with the use of a variety of techniques.
  • a biological sample of the subject containing either
  • DNA or RNA derived from the subject is assayed for the presence of a mutant 15 kDa selenoprotein gene.
  • Suitable biological samples include samples containing genomic DNA or RNA obtained from body cells, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen.
  • the assay may be performed on cDNA made from mRNA obtained from a biological sample
  • the detection of mutations in the 15 kDa selenoprotein gene may be detected using single-strand conformational polymo ⁇ hism (SSCP) analysis
  • SSCP single-strand conformational polymo ⁇ hism
  • the detection in the biological sample of either a mutant 15 kDa selenoprotein gene or a mutant 15 kDa selenoprotein RNA may also be performed by a number of other methodologies known in the art, as outlined below
  • the presence of the polymo ⁇ hic form C81 1/G1125 may be detected by such means
  • a method for detecting a polymo ⁇ hism in a human 15 kDa selenoprotein gene, cDNA or RNA in a biological sample comprises hybridizing the sample with a nucleic acid probe under conditions whereby the probe will hybridize to 15 kDa selenoprotein gene, cDNA or RNA carrying a specified particular polymo ⁇ hism such as T81 1 Al 125 or T81 1/A1 125, but not to the other polymo ⁇ hism of the 15 kDa selenoprotein gene, cDNA or RNA (C811/G1125)
  • the human "wild-type" sequence is considered to be that shown in Seq I D No 2
  • RT-PCR reverse transcribed RNA
  • DNA extracted from lymphocytes or other cells may be used directly for amplification
  • the direct amplification from genomic DNA would be appropriate for analysis of the entire 15 kDa selenoprotein gene including regulatory sequences located upstream and downstream from the open reading frame Reviews of direct DNA diagnosis have been presented by Caskey (Science 236 1223-8, 1989) and by Landegren et al (Science 242 229-37, 1989)
  • the detection of specific DNA mutations may be achieved by methods such as hybridization using specific oligonucleotides (Wallace et al, 1986, Cold Spring Harbor Symp. Quant. Biol. 51 :257-61), direct DNA sequencing (Church and Gilbert, 1988, Proc. Natl. Acad. Sci. USA 81 : 1991-5), the use of restriction enzymes (Flavell et al, 1978, Cell 15:25; Geever et al, 1981 , Proc. Natl. Acad. Sci USA 78:5081), discrimination on the basis of electrophoretic mobility in gels with denaturing reagent (Myers and Maniatis, 1986, Cold Spring Harbor Symp. Quant. Biol.
  • oligonucleotides specific to normal or mutant sequences may be chemically synthesized using commercially available machines, labelled radioactively with isotopes (such as 32 P) or non-radioactively with tags such as biotin (Ward and Langer, 1981 , Proc. Natl. Acad. Sci. USA 78:6633-57).
  • Sequence differences between normal and mutant forms of that gene may also be revealed by the direct DNA sequencing method of Church and Gilbert (Proc. Natl. Acad. Sci. USA 81 : 1991 -5, 1988). Cloned DNA segments may be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR (Wrichnik et al, 1987, Nucleic Acids Res. 15:529-42; Wong et al, 1987, Nature 330:384-6; Stoflet et al, 1988, Science 239:491-4). In this approach, a sequencing primer which lies within the amplified sequence is used with double-stranded PCR product or single-stranded template generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent tags.
  • Sequence alterations may occasionally generate fortuitous restriction enzyme recognition sites or may eliminate existing restriction sites. Changes in restriction sites are revealed by the use of appropriate enzyme digestion followed by conventional gel-blot hybridization (Southern, 1975, J. Mol. Biol. 98:503). DNA fragments carrying the site (either normal or mutant) are detected by their reduction in size or increase of corresponding restriction fragment numbers. Genomic DNA samples may also be amplified by PCR prior to treatment with the appropriate restriction enzyme; fragments of different sizes are then visualized under UV light in the presence of ethidium bromide after gel electrophoresis. Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing reagent. Small sequence deletions and insertions can be visualized by high-resolution gel electrophoresis. For -35-
  • PCR product with small deletions is clearly distinguishable from a normal sequence on an 8%> non-denaturing polyacrylamide gel (Nagamine et al, 1989, Am. J. Hum. Genet. 45:337-9).
  • DNA fragments of different sequence compositions may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific "partial-melting" temperatures (Myers et al, 1985, Science
  • a method of detecting a mutation comprising a single base substitution or other small change could be based on differential primer length in a PCR.
  • an invariant primer could be used in addition to a primer specific for a mutation.
  • the PCR products of the normal and mutant genes can then be differentially detected in acrylamide gels.
  • DNA fragments may also be visualized by methods where the individual DNA samples are not immobilized on membranes.
  • the probe and target sequences may be both in solution, or the probe sequence may be immobilized (Saiki et al, 1989, Proc. Nat. Acad. Sci. USA 86:6230-4).
  • a variety of detection methods such as autoradiography involving radioisotopes, direct detection of radioactive decay (in the presence or absence of scintillant), spectrophotometry involving calorigenic reactions and fluorometry involved fluorogenic reactions, may be used to identify specific individual genotypes.
  • a system capable of detecting such multiple mutations would be desirable.
  • a PCR with multiple, specific oligonucleotide primers and hybridization probes may be used to identify all possible mutations at the same time (Chamberlain et al, 1988, Nucl. Acids Res. 16:1141-55).
  • the procedure may involve immobilized sequence-specific oligonucleotides probes (Saiki et al. 1989, Proc. Nat. Acad. Sci. USA 86:6230-4).
  • compositions of the present invention including 15 kDa selenoprotein-specific antibodies and nucleic acid probes and primers, may be used to detect and/or quantify the level of 15 kDa selenoprotein polypeptide or mRNA in a biological sample.
  • Biological samples suitable for analysis include biopsy samples, such as tumor biopsies, and biological fluids containing cellular material, such as blood, cerebrospinal fluid and saliva.
  • Determining and/or quantifying the levels of 15 kD selenoprotein polypeptide and mRNA would be useful for detecting reduced levels of the 15 kDa selenoprotein and mRNA which result from, for example, mutations in the promoter regions of the 15 kDa selenoprotein gene or mutations -36-
  • such determinations may provide valuable information about the ability of the cell to inco ⁇ orate selenium into proteins, as well as information about oxidative stress.
  • Abnormally low levels of 15 kDa selenoprotein polypeptide or mRNA may be indicative of the presence of cancer; such measurements may also be useful to measure the efficacy of cancer treatment.
  • the determination of reduced 15 kDa selenoprotein polypeptide or mRNA levels would be an alternative or supplemental approach to the direct determination of a patient's status by nucleotide sequence determination outlined above.
  • the availability of antibodies specific to the 15 kDa selenoprotein polypeptide allows the quantitation of cellular 15 kDa selenoprotein polypeptide by one of a number of immunoassay methods which are well known in the art and are presented in Harlow and Lane (Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York. 1988). Such methods include antibody capture assays, antigen capture assays and two antigen sandwich assays. For certain assays, a detectable label may be conjugated to the antibody.
  • Suitable detectable labels include radioactive labels, fluorescent labels and enzymes. Detection and quantification of 15 kDa selenoprotein mRNA levels in a biological sample may be achieved using the probes and primers described above in conjunction with standard laboratory techniques, including quantitative RT-PCR and Northern blotting.
  • a significant (preferably 50% or greater) reduction in the amount of 15 kDa selenoprotein polypeptide in the cells of a subject compared to the amount of 15 kDa selenoprotein polypeptide found in control ("healthy") cells would be taken as an indication that the subject may be suffering from, or at risk from, cancer.
  • kits suitable for the detection and quantification of 15 kDa selenoprotein polypeptide or mRNA in biological specimens comprise a container holding a 15 kDa selenoprotein polypeptide-specific binding agent, such as a monoclonal antibody.
  • the antibody may be bound to a solid substrate, such as a column or microtiter plate well.
  • the kit may further include a second specific binding agent that specifically binds to either the 15 kDa selenoprotein polypeptide, or the first specific binding agent.
  • the second specific binding agent may be conjugated with a label molecule that facilitates detection of the second agent when bound to its target.
  • kits suitable for detecting or quantifying the 15 kDa selenoprotein mRNA comprise a container holding one of more nucleic acid primers or probes as provided above.
  • the nucleic acid probes may be conjugated to a suitable label molecule that facilitates detection of the probe when bound to its target.
  • suitable label molecules are known in the art and include radionuclides and biotin.
  • the invention also provides methods for screening compounds for their ability to inhibit, facilitate or modulate the expression of 15 kDa selenoprotein polypeptide and mRNA molecules, for use in the in vitro screening of novel agonist and antagonist compounds.
  • Such compounds include candidate cancer therapeutics, such as candidate prostate cancer therapeutics.
  • candidate cancer therapeutics such as candidate prostate cancer therapeutics.
  • methods comprise measuring 15 kDa selenoprotein polypeptide or mRNA levels in mammalian cells, treating those cells with the candidate agent, and then measuring the 15 kDa selenoprotein polypeptide or mRNA levels to determine what effect, if any, the agent had on expression.
  • the cells to which the candidate agent is administered may be cultured in vitro.
  • Suitable cell lines include human tumor and non-tumor cell lines available from public collections, such as ATCC (Manassas, VA).
  • ATCC Manassas, VA
  • the testing of such candidate agents may be performed in laboratory animals, such as mice or rats.
  • Measurement of the levels of 15 kDa selenoprotein polypeptide or mRNA in this latter situation may be accomplished by analysis of biopsy samples or cells from bodily fluids, such as blood.
  • the levels of 15 kDa selenoprotein polypeptide and/or mRNA may be performed using reagents and methods described above.
  • transgenic non-human animal models over-expressing the 15 kDa selenoprotein polypeptide, or variant or mutated versions of the polypeptide are useful for the assessment of agents, such as agonists and antagonists of the polypeptide.
  • the mouse 15 kDa selenoprotein polypeptide cDNA may be employed in conjunction with known methodologies for creating transgenic mice that over-express an introduced nucleic acid sequence to produce useful animal -38-
  • transgenic animal models Suitable techniques for generating such transgenic animal models include those described in U.S. Patent Nos. 5,489,742 ("Transgenic rats and animal models of inflammatory disease"), 5,489,743 ("Transgenic animal models for fhrombocytopenia"), 5,304,489 ("DNA sequences to target proteins to the mammary gland for efficient secretion"), 5,476,995 (“Peptide production”), and 5,487,992 ("Cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same”), and references cited therein.
  • the relationship between the 15 kDa selenoprotein may be further explored by the creation of double transgenic mice, transgenic for oncogene sequences as well as nucleic acids that encode the 15 kDa selenoprotein.
  • nucleic acids encoding the 15 kDa selenoprotein may be introduced into tumor cells, which cells may then be used to study tumorigenesis in laboratory animal models, such as mice.
  • conditional gene silencing can be used to generate transgenic mice (for reviews see Porter, 1998, Trends Genetics, vol. 14; Rajewsky et al, 1996, J Clin. Invest 98:S51-S53).
  • Conditional silencing of a gene allows cells to accumulate prior to the inactivation (functional deletion) of the gene. This approach is advantageous for several reasons. If the gene of interest is an essential gene, mutations in that gene might be lethal, leaving no mouse to study gene function.
  • this method allows one to generate models of somatically acquired genetic diseases, such as most forms of cancer, rather than of inherited ones.
  • the strategy of this method utilizes the bacteriophage-derived Cre-lox system.
  • the Cre enzyme recognizes a sequence motif of 34 bp, called loxP. If a DNA segment is flanked by two loxP sites in the same orientation, Cre excies that segment from the DNA, leaving a single loxP site behind. Conditional targeting is accomplished by crossing responder mice, carrying the loxP flanked target gene, with regulator mice carrying the Cre transgene, which is expressed in a cell-type-specific or inducible manner.
  • the present invention describes for the first time the existence of the 15 kDa selenoprotein, provides evidence of a link between low levels of this protein and cancer, and provides methods for determining levels of the 15 kDa selenoprotein. Supplementation of the diet with selenium represents one way in which the level of the 15 kDa selenoprotein may be enhanced, with the goal of reducing susceptibility to cancer in patients with a predetermined genetic susceptibility.
  • the present invention provides a method for enhancing the level of the 15 kDa selenoprotein in a mammal, by administering to the mammal a dietary selenium supplement.
  • the method involves a prior determination that the level of 15 kDa selenoprotein in the mammal is lower than the measured average for such mammals.
  • the invention provides a method for dietary regulation in which the level of 15 kDa selenoprotein in the cells of a mammal is -39-
  • selenium supplementation can take the form of an oral supplement, such as the oral administration of 200 ⁇ g of selenuium per day, as described by Clark et al. (JAMA, 276: 1957-63, 1996)
  • the present invention relates to a method of treating tumors by overexpressing the 15 kDa selenoprotein in cells which have an abnormally low amount of the 15 kDa selenoprotein, or in the cells of a patient having a higher risk for cancers associated with low-levels of 15 kDa selenoprotein.
  • These methods may be accomplished by introducing a gene coding for the 15 kDa selenoprotein (or a variant thereof) into the person.
  • a general strategy for transferring genes into donor cells is disclosed in U.S. Patent No. 5,529,774.
  • a gene encoding a protein having therapeutically desired effects is cloned into a viral expression vector, and that vector is then introduced into the target organism. The virus infects the cells, and produces the protein sequence in vivo, where it has its desired therapeutic effect. See, for example, Zabner et al. (Cell 75:207-16, 1993).
  • the genetic or protein elements may only be necessary to introduce the genetic or protein elements into certain cells or tissues.
  • introducing them into only the skin may be sufficient.
  • the nucleic acid sequence encoding at least one therapeutic agent is under the control of a suitable promoter.
  • suitable promoters which may be employed include, but are not limited to, the gene's native promoter, retroviral LTR promoter, or adenoviral promoters, such as the adenoviral major late promoter: the cytomegalovirus (CMV) promoter; the Rous Sarcoma Virus (RSV) promoter; inducible promoters, such as the MMTV promoter; the metallothionein promoter; heat shock promoters; the albumin promoter; the histone promoter; the ⁇ -actin promoter; TK promoters; B19 parvovirus promoters; and the ApoAI promoter.
  • CMV cytomegalovirus
  • RSV Rous Sarcoma Virus
  • inducible promoters such as the MMTV promoter
  • the metallothionein promoter such as the MMTV promoter
  • heat shock promoters such as the
  • the recombinant nucleic acid can be administered to the animal host by any method which allows the recombinant nucleic acid to reach the appropriate cells. These methods include injection, infusion, deposition, implantation, or topical administration. Injections can be intradermal or subcutaneous.
  • the recombinant nucleic acid can be delivered as part of a viral vector, such as avipox viruses, recombinant vaccinia virus, replication-deficient adenovirus strains or poliovirus, or as a non- infectious form such as naked DNA or liposome encapsulated DNA. -40-
  • Adenoviral vectors may include essentially the complete adenoviral genome (Shenk et al, Curr. Top. Microbiol. Immunol. 111:1-39, 1984).
  • the adenoviral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted.
  • the vector includes an adenoviral 5' ITR (inverted terminal repeats); an adenoviral 3' ITR; an adenoviral encapsidation signal; a DNA sequence encoding a therapeutic agent; and a promoter for expressing the DNA sequence encoding a therapeutic agent.
  • the vector is free of at least the majority of adenoviral El and E3 DNA sequences, but is not necessarily free of all of the E2 and E4 DNA sequences, and DNA sequences encoding adenoviral proteins transcribed by the adenoviral major late promoter.
  • the vector may be an adeno-associated virus (AAV) such as described in U.S. Patent No. 4,797,368 (Carter et al.) and AAV type 4 (Chiorini et al. J. Virol. 71 :6823-33, 1997) and AAV type 5 (Chiorini et al. J. Virol. 73: 1309-19, 1999)
  • AAV adeno-associated virus
  • Such a vector may be constructed according to standard techniques, using a shuttle plasmid which contains, beginning at the 5' end, an adenoviral 5' ITR, an adenoviral encapsidation signal, and an El a enhancer sequence; a promoter (which may be an adenoviral promoter or a foreign promoter); a tripartite leader sequence, a multiple cloning site (which may be as herein described); a poly A signal; and a DNA segment which corresponds to a segment of the adenoviral genome.
  • the DNA segment serves as a substrate for homologous recombination with a modified or mutated adenovirus.
  • the plasmid may also include a selectable marker and an origin of replication.
  • the origin of replication may be a bacterial origin of replication.
  • a desired DNA sequence encoding a therapeutic agent may be inserted into the multiple cloning site of the plasmid.
  • the plasmid may be used to produce an adenoviral vector by homologous recombination with a modified or mutated adenovirus in which at least the majority of the El and E3 adenoviral DNA sequences have been deleted. Homologous recombination may be effected through co- transfection of the plasmid vector and the modified adenovirus into a helper cell line, such as 293 cells, by CaP0 4 precipitation.
  • a helper cell line such as 293 cells
  • the homologous recombination produces a recombinant adenoviral vector which includes DNA sequences derived from the shuttle plasmid between the Not I site and the homologous recombination fragment, and DNA derived from the El and E3 deleted adenovirus between the homologous recombination fragment and the 3' ITR.
  • the adenovirus may be constructed by using a yeast artificial chromosome (or YAC) containing an adenoviral genome according to the method described in Ketner et al. (Proc. Natl. Acad. Sci., USA , 91 :6186-90, 1994), in conjunction with the teachings contained herein.
  • the adenovirus yeast artificial chromosome is produced by homologous recombination in vivo between adenoviral DNA and yeast artificial chromosome plasmid vectors carrying segments of the adenoviral left and right genomic termini.
  • therapeutic agent then may be cloned into the adenoviral DNA.
  • the modified adenoviral genome then is excised from the adenovirus yeast artificial chromosome in order to be used to generate adenoviral vector particles as hereinabove described.
  • the adenoviral particles are administered in an amount effective to produce a therapeutic effect in a host.
  • the exact dosage of adenoviral particles to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient to be treated, and the nature and extent of the disease or disorder to be treated.
  • the adenoviral particles may be administered as part of a preparation having a titer of adenoviral particles of at least 1 x 10 10 pfu/ml, and in general not exceeding 2 x 10" pfu/ml.
  • the adenoviral particles may be administered in combination with a pharmaceutically acceptable carrier in a volume up to 10 ml.
  • the pharmaceutically acceptable carrier may be, for example, a liquid carrier such as a saline solution, protamine sulfate (Elkins-Sinn,, Inc., Cherry Hill, N.J.), or Polybrene (Sigma Chemical).
  • the viral vector is a retroviral vector.
  • retroviral vectors which may be employed include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.
  • the vector is generally a replication defective retrovirus particle.
  • Retroviral vectors are useful as agents to effect retroviral-mediated gene transfer into eukaryotic cells.
  • Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e, gag, pol, and env). are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal. New genes may be inco ⁇ orated into proviral backbones in several general ways.
  • Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter. Alternatively, two genes may be expressed from a single promoter by the use of an Internal Ribosome Entry Site.

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Abstract

La présente invention concerne une protéine de 15 kDa contenant du sélénium ('sélénoprotéine'). Cette protéine s'exprime de manière différentielle dans des cellules cancéreuses, telles que des cellules du cancer de la prostate. Une corrélation existe entre la présence d'un polymorphisme aux positions nucléotidiques 811 et 1125 du gène de la sélénoprotéine de 15 kDa et la présence d'un cancer. Ce polymorphisme est plus courant dans la population afro-américaine. La détermination d'un génotype individuel peut être utilisée comme indicateur du besoin d'une supplémentation alimentaire en sélénium de façon à inhiber un développement de tumeur. La présente invention concerne aussi des compositions qui comprennent la protéine isolée, des agents liants spécifiques qui reconnaissent la protéine, des séquences d'acides nucléiques sous-jacentes, et des méthodes d'utilisations de telles compositions.
PCT/US1999/007560 1998-04-06 1999-04-06 Selenoproteine mammalienne montrant une expression differentielle dans des cellules tumorales WO1999051637A1 (fr)

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US10/919,554 US7442543B2 (en) 1998-04-06 2004-08-16 Mammalian selenoprotein differentially expressed in tumor cells
US12/234,968 US7776607B2 (en) 1998-04-06 2008-09-22 Mammalian selenoprotein differentially expressed in tumor cells
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US7442543B2 (en) * 1998-04-06 2008-10-28 The United States Of America As Represented By The Department Of Health And Human Services Mammalian selenoprotein differentially expressed in tumor cells
US7776607B2 (en) 1998-04-06 2010-08-17 The United States Of America As Represented By The Department Of Health And Human Services Mammalian selenoprotein differentially expressed in tumor cells
WO2001012657A3 (fr) * 1999-08-16 2001-08-23 Karolinska Innovations Ab Procedes et moyens destines a l'expression de la selenoproteine
WO2002050274A3 (fr) * 2000-12-18 2003-08-07 Zymogenetics Inc Proteine zsel1 contenant de la selenocysteine
RU2185819C1 (ru) * 2001-10-18 2002-07-27 Дорофеенко Алла Ивановна Средство, обладающее противоопухолевым действием
WO2012125051A1 (fr) 2011-03-14 2012-09-20 Pomorski Uniwersytet Medyczny Génotypes de sélénoprotéines et teneur en sélénium du sérum en tant que marqueurs du risque de cancer
JP2015513912A (ja) * 2012-04-02 2015-05-18 モデルナ セラピューティクス インコーポレイテッドＭｏｄｅｒｎａ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． タンパク質の産生のための修飾ポリヌクレオチド
US9814760B2 (en) 2012-04-02 2017-11-14 Modernatx, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9827332B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of proteins
US9828416B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
JP2019107022A (ja) * 2012-04-02 2019-07-04 モデルナティエックス インコーポレイテッドＭｏｄｅｒｎａＴＸ，Ｉｎｃ． タンパク質の産生のための修飾ポリヌクレオチド
US10577403B2 (en) 2012-04-02 2020-03-03 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US10703789B2 (en) 2012-04-02 2020-07-07 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
JP2021045163A (ja) * 2012-04-02 2021-03-25 モデルナティエックス インコーポレイテッドＭｏｄｅｒｎａＴＸ，Ｉｎｃ． タンパク質の産生のための修飾ポリヌクレオチド

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