EP0804546A1 - Reductase specifique de la prostate chez l'homme - Google Patents

Reductase specifique de la prostate chez l'homme

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Publication number
EP0804546A1
EP0804546A1 EP95911712A EP95911712A EP0804546A1 EP 0804546 A1 EP0804546 A1 EP 0804546A1 EP 95911712 A EP95911712 A EP 95911712A EP 95911712 A EP95911712 A EP 95911712A EP 0804546 A1 EP0804546 A1 EP 0804546A1
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EP
European Patent Office
Prior art keywords
polypeptide
polynucleotide
psr
sequence
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP95911712A
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German (de)
English (en)
Other versions
EP0804546A4 (fr
Inventor
Wei Wu He
Paul S. Meissner
Peter L. Hudson
Craig A. Rosen
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Human Genome Sciences Inc
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Human Genome Sciences Inc
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Publication date
Application filed by Human Genome Sciences Inc filed Critical Human Genome Sciences Inc
Publication of EP0804546A1 publication Critical patent/EP0804546A1/fr
Publication of EP0804546A4 publication Critical patent/EP0804546A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the prostatic specific reductase genes and polypeptides of the present invention.
  • a nucleic acid sequence in a sample derived from a tissue other than the prostate is amplified and detected.
  • the sample contains a nucleic acid or a mixture of nucleic acids, at least one of which is suspected of containing the sequence coding for PSR polypeptide.
  • initially RNA is isolated from the cells.
  • a competition assay may be employed where an antibody specific to PSR proteins are attached to a solid support and labeled PSR proteins and a sample derived from the host are passed over the solid support and the amount of label detected, for example, by liquid scintillation chromatography, can be correlated to a quantity of PSR proteins in the sample.
  • Antibodies of this type may also be used to do in vivo imaging, for example, by labeling the antibodies to facilitate scanning of the pelvic area and the prostate.
  • One method for imaging comprises contacting any tumor cells of the prostate to be imaged with an anti-PSR antibody labeled with a detectable marker. The method is performed under conditions such that the labeled antibody binds to the PSR.
  • the antibodies interact with the prostate, for example, prostate cancer cells, and fluoresce upon such contact such that imaging and visibility of the prostate is enhanced to allow a determination of the diseased or non-diseased state of the prostate.
  • the prostatic specific reductase of this invention was identified 3 times in the stage B2 human prostate cancer library and 7 times in the stage C prostate cancer library, and most notably was not identified at all in the normal human prostate library or from libraries derived from non-prostate tissues, indicating its importance as a marker for prostate disorders.
  • the polynucleotide which encodes for the mature polypeptide of SEQ ID No. 2 or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide or the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non- coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide or which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of SEQ ID No. 2 or the polypeptide encoded by the cDNA of the deposited clone.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
  • a marker sequence is a hexa-histidine tag which may be supplied by a vector, preferably a pQE-9 vector, which provides for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • HA hemagglutinin
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984) ) .
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • the present invention further relates to a PSR polypeptide which has the deduced amino acid sequence of SEQ ID No. 2 or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) .
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the PSR genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those of ordinarily skill in the art.
  • the polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques.
  • the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus,* yeast plasmids,- vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoter for example, LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • bacterial cells such as E. coli. Streptomvces, Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Sf9: animal cells such as CHO, COS or Bowes melanoma
  • adenoviruses plant cells, etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) .
  • any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation (Davis, L., Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)) .
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al. , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenoviru ⁇ enhancers.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , ⁇ -factor, acid phosphatase, or heat shock proteins, among others.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella tvphimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcu ⁇ , although others may also be employed as a matter of choice.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) .
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA) .
  • pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • the PSR polypeptide can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) .
  • a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
  • the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may also include an initial methionine amino acid residue.
  • assays which may be used to screen for therapeutics to inhibit PSR, since PSR is a reductase and may be necessary for the proliferation of the prostate cancer cells.
  • the present invention discloses methods for selecting a therapeutic which forms a complex with PSR with sufficient affinity to prevent the biological action of PSR.
  • the methods include various assays, including competitive assays where the PSR is immobilized to a support, and is contacted with a natural substrate for PSR and a labeled therapeutic either simultaneously or in either consecutive order, and determining whether the therapeutic effectively competes with the natural substrate in a manner sufficient to prevent binding of PSR to its substrate.
  • the natural substrate is labeled and the therapeutic is unlabeled.
  • the substrate is immobilized to a support, and is contacted with both labeled PSR and a therapeutic (or unlabeled PSR and a labeled therapeutic) , and it is determined whether the amount of PSR bound to the substrate is reduced in comparison to the assay without the therapeutic added.
  • the PSR may be labeled with the anti-PSR antibodies of the subject invention.
  • elements which undergo simultaneous oxidation and reduction for example hydrogen and oxygen which together form water
  • radioactivity e.g., tritium
  • Potential antagonists to PSR including antibody, i.e., an anti-idiotypic antibody as described above, or in some cases, an oligonucleotide, which binds to the polypeptide.
  • Another potential PSR antagonist is an antisense construct prepared using antisense technology.
  • Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide sequence which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PSR polypeptide (antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of PSR.
  • Fragments of the full length PSR gene may be used as a hybridization probe for a cDNA library to isolate the full length PSR gene and to isolate other genes which have a high sequence similarity to the PSR gene or similar biological activity.
  • Probes of this type can be, for example, between 20 and 2000 base pairs. Preferably, however, the probes have between 30 and 50 bases.
  • the probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete PSR gene including regulatory and promotor regions, exons, and introns.
  • An example of a screen comprises isolating the coding region of the PSR gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotide ⁇ having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridize
  • the PSR polypeptides or agonists or antagonists may be employed in combination with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the pharmaceutical compositions may be employed in conjunction with other therapeutic compounds.
  • the pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intra-anal or intradermal routes.
  • the pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 ⁇ g/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
  • PSR polypeptides and agonists and antagonists which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy.”
  • cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time.
  • Verma et al. Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988) .
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb) .
  • polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • typically l ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume.
  • Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37"c are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
  • Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980).
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • venous blood is obtained with a standard venipuncture technique using heparinized tubes.
  • Whole blood is mixed with an equal volume of phosphate buffered saline, which is then layered over 8 ml of Ficoll (Pharmacia, Uppsala, Sweden) in a 15-ml polystyrene tube.
  • the gradient is centrifuged at 1800 X g for 20 min at 5°C.
  • lymphocyte and granulocyte layer (approximately 5 ml) is carefully aspirated and rediluted up to 50 ml with phosphate-buffered saline in a 50-ml tube, which is centrifuged again at 1800 X g for 20 min. at 5°C. The supernatant is discarded and the pellet containing nucleated cells is used for RNA extraction using the RNazole B method as described by the manufacturer (Tel-Test Inc., Friendswood, TX) .
  • oligonucleotide primers are employed to amplify the PSR nucleotide sequence present in the sample: the 5' primer is 5' AAGAGATCCAGACCACGACAGG 3' (SEQ ID No. 3) and the 3' primer is 5' AAGGCACAGTGCAGCCTGGTCT 3' (SEQ ID No. 4).
  • the reverse transcriptase reaction and PCR amplification are performed sequentially without interruption in a Perkin Elmer 9600 PCR machine (Emeryville, CA) .
  • Four hundred ng total RNA in 20 ⁇ l diethylpyrocarbonate-treated water are placed in a 65°C water bath for 5 min. and then quickly chilled on ice immediately prior to the addition of PCR reagents.
  • cycle 3 is 95°C for 1 min. 60°C for 1 min., and 72°C for 1 min. (10 cycles); cycle 4 is 95°C for 1 min., 60°C for 1 min., and 72°C for 2 min. (8 cycles) ; cycle 5 is 72°C for 15 min. (1 cycle) ; and the final cycle is a 4°C hold until sample is taken out of the machine.
  • the 50- ⁇ l PCR products are concentrated down to 10 ⁇ l with vacuum centrifugation, and the entire sample is then run on a thin 1.2 % Tris-borate- EDTA agarose gel containing ethidium bromide.
  • a 1.2 kb band indicates that genomic DNA is amplified, which is not indicative of prostate cancer metastases. However, if the band is somewhat smaller, a 567 base pair product, the indication is that the PSR mRNA is amplified by PCR and cells, other than the prostate, are activley transcribing PSR protein and are circulating in the blood, i.e. metasti ⁇ izing. All specimens are analyzed at least twice to confirm a positive or negative outcome.
  • Verification of the nucleotide sequence of the PCR products is done by microsequencing.
  • the PCR product is purified with a Qiagen PCR Product Purification Kit (Qiagen, Chatsworth, CA) as described by the manufacturer.
  • Qiagen Quality of Service
  • Chatsworth, CA Quality of Service
  • One ⁇ g of the PCR product undergoes PCR sequencing by using the Taq DyeDeoxy Terminator Cycle sequencing kit in a Perkin-Elmer 9600 PCR machine as described by Applied Biosystems (Foster, CA) .
  • the sequenced product is purified using Centri-Sep columns (Princeton Separations, Adelphia, NJ) as described by the company. This product is then analyzed with an ABI model 373A DNA sequencing system (Applied Biosystems) integrated with a Macintosh Ilci computer.
  • pQE-9 is then digested with Sail and Xbal.
  • the amplified sequences are ligated into pQE-9 and are inserted in frame with the sequence encoding for the histidine tag and the RBS.
  • the ligation mixture is then used to transform E. coli strain ml5/pREP4 available from Qiagen under the trademark M15/rep 4 by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989) .
  • M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kanamycin resistance (Kan r ) .
  • PSR 90% pure is eluted from the column in 6 molar guanidine HC1 pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HC1, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.
  • Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.
  • COS cells are transfected with the expre ⁇ sion vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)) .
  • the expression of the PSR HA protein is detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)) .
  • Cells are labelled for 8 hours with 35 S-cysteine two days post transfection.

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Abstract

Cette invention concerne un polypeptide de réductase spécifique de la prostate chez l'homme, des polynucléotides codant pour ce polypeptide ainsi qu'un procédé de fabrication de ce polypeptide par des techniques de recombinaison. Cette invention concerne également des procédés d'utilisation de ces polynucléotides en tant qu'indicateur de diagnostic du cancer de la prostate et en tant qu'agent indiquant si le cancer de la prostate est métastasé. La présente invention fait aussi état d'anticorps spécifiques au polypeptide de réductase spécifique de la prostate, lesquels peuvent servir à cibler les cellules du cancer de la prostate et peuvent être employés en tant qu'élément entrant dans la composition d'un vaccin contre ce cancer. Cette invention décrit enfin des procédés de criblage d'agonistes et d'antagonistes du polypeptide, ainsi que des emplois thérapeutiques de ces antagonistes.
EP95911712A 1995-01-20 1995-01-20 Reductase specifique de la prostate chez l'homme Withdrawn EP0804546A4 (fr)

Applications Claiming Priority (1)

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PCT/US1995/001827 WO1996022360A1 (fr) 1995-01-20 1995-01-20 Reductase specifique de la prostate chez l'homme

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EP0804546A1 true EP0804546A1 (fr) 1997-11-05
EP0804546A4 EP0804546A4 (fr) 2000-09-20

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JP (1) JPH10513044A (fr)
AU (1) AU1918095A (fr)
WO (1) WO1996022360A1 (fr)

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EP1692936A1 (fr) 2000-10-03 2006-08-23 GIE-CERBM, Centre Europeen de Recherche en Biologie et en Médecine Méthode utilisant la protéine de fusion Cre-ERT2 pour la recombinaison dirigée conditionnelle de l'ADN chez la souris

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US6287854B1 (en) 1996-10-22 2001-09-11 Imperial Cancer Research Technology Limited Diagnosis of susceptibility to cancer and treatment thereof
US7081514B2 (en) 1998-09-01 2006-07-25 Genentech, Inc. PRO1347 polypeptides
US6156515A (en) 1999-02-09 2000-12-05 Urocor, Inc. Prostate-specific gene for diagnosis, prognosis and management of prostate cancer
AU4484400A (en) * 1999-04-23 2000-11-10 University Of Washington Prostate-specific polynucleotides, polypeptides and their methods of use
AU2001274981A1 (en) * 2000-05-25 2001-12-03 Incyte Genomics, Inc. Drug metabolizing enzymes
GB0810097D0 (en) 2008-06-03 2008-07-09 Magnomatics Ltd Magnetic gear
WO2010101528A1 (fr) * 2009-03-02 2010-09-10 Agency For Science, Technology And Research Biomarqueur et traitement pour le cancer

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Title
AKALU A ET AL: "Somatic mutations at the SRD5A2 locus encoding prostatic steroid 5alpha-reductase during prostate cancer progression." JOURNAL OF UROLOGY, (1999 APR) 161 (4) 1355-8., XP000881743 *
Andersson et al., Nature 354:159-161 (1991) *
See also references of WO9622360A1 *
THIGPEN A E ET AL: "Molecular genetics of steroid 5 alpha-reductase 2 deficiency." JOURNAL OF CLINICAL INVESTIGATION, (1992 SEP) 90 (3) 799-809., XP000087278 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1692936A1 (fr) 2000-10-03 2006-08-23 GIE-CERBM, Centre Europeen de Recherche en Biologie et en Médecine Méthode utilisant la protéine de fusion Cre-ERT2 pour la recombinaison dirigée conditionnelle de l'ADN chez la souris

Also Published As

Publication number Publication date
AU1918095A (en) 1996-08-07
WO1996022360A1 (fr) 1996-07-25
EP0804546A4 (fr) 2000-09-20
JPH10513044A (ja) 1998-12-15

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