WO1995025178A1 - Methods for detecting 12-lipoxygenase and its encoding dna - Google Patents

Methods for detecting 12-lipoxygenase and its encoding dna Download PDF

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WO1995025178A1
WO1995025178A1 PCT/US1995/001623 US9501623W WO9525178A1 WO 1995025178 A1 WO1995025178 A1 WO 1995025178A1 US 9501623 W US9501623 W US 9501623W WO 9525178 A1 WO9525178 A1 WO 9525178A1
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rna
lipoxygenase
dna
cdna
seq
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PCT/US1995/001623
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French (fr)
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Kenneth V. Honn
Yong Q. Chen
John D. Crissman
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Biomide Investment Limited Partnership
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90241—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Definitions

  • the present invention relates to methods for detecting 12-lipoxygenase or the DNA encoding this enzyme.
  • the present invention relates to a method using antibody or DNA-DNA probe binding for the detection.
  • the present invention also relates to the use of a polymerase chain reaction to produce a cDNA encoding a unique part of 12-lipoxygenase which is detected.
  • Metastasis is a cascade which requires multiple factors expressed by tumor cells as well as surrounding stromal cells, and is subject to distinct stimulus regulations (Hart, I. R. , et al., Biocim. Biophys. Acta. 989:65-84, (1989)) .
  • tumor cells undergo extensive cell- cell and cell-matrix interactions.
  • Two cell types, i.e., platelets and endothelial cells with which tumor cells interact during hematogenous metastasis are capable of producing a vast array of lipid mediators by either direct or transcellular metabolism of precursors (Chen, Y. Q., et al., Cancer Metast. Rev. 11, 389-410 (1992a)).
  • Platelets are capable of converting arachidonic acid (AA) to prostaglandins and predominantly to thromboxane A 2 through the cyclooxygenase (COX) pathway. Platelets also metabolize AA through the lipoxygenase (LOX) pathway producing 12 (S) -hydroxyeicosatetraenoic acid (12(S)-HETE) (Moncada, S., and Higgs, E. A., Clin. Heamato. 15: 273- 292 (1986)). After intravasation, tumor cells interact with platelets and induce platelet aggregation with concomitant synthesis of platelet 12(S)-HETE (Honn, K. V., et al., Biochem. Biophys. Res.
  • LOX lipoxygenase
  • platelets can be a contributor of 12(S)-HETE to facilitate tumor cell interaction with endothelial cells and subendothelial matrix.
  • 12(S)-HETE increases tumor cell adhesion to endothelium (Honn, K. V., et al., Biochem. Biophys. Res. Commun. 29:384-389 (1987) ; Grossi, I. M.
  • Tumor cells express a variety of adhesion molecules.
  • integin ⁇ ⁇ rb jS 3 is an important receptor for tumor cell-host cell and tumor cell-matrix interaction (Chen, Y. Q. , et al., J. Biol. Chem. 267, 17314-17320 (1992b); Chang, . C. , et al., Int. J. Cancer 51, 445-451, (1992a)).
  • Pretreatment of tumor cells with exogenous 12(S)-HETE enhances their adhesion to and spreading on fibronectin, which is mediated exclusively by ⁇ 3 receptors and in a cytoskeleton-dependent manner (Chopra, H., et al., Int. J.
  • 12(S)-HETE also appears to regulate other factors which are important to tumor cell metastasis.
  • Pretreatment of tumor cells with 12(S)-HETE stimulates cell motility (Raz, A., et al., Kluwer Academic Publishers, pp645-649 (1992) ; Timar, et al, Intl. J. Cancer 55:1003-1010 (1993)) and release of cathepsin B (Sloane, B. F.
  • 12(S)-HETE increases tumor cell motility and release of protease.
  • 12(S)-HETE augments the rate of tumor cell extravasation and finally metastasis.
  • 12(S)-HETE has significant importance in hematogenous metastasis.
  • Figure 1 is an electrophoresis gel showing 12- lipoxygenase expression in various types of tumor cells.
  • Total RNA from each cell line was reverse transcribed and amplified by polymerase chain reaction (RT-PCR) . Ten ⁇ l of the final PCR products were separated on 2% agarose gel. Control RT-PCR was run under the same conditions without total RNA.
  • ⁇ EcoRV ⁇ DNA EcoRV fragment as marker
  • H Platelet human platelet
  • R Platelet rat platelet
  • M Platelet mouse platelet.
  • Tumor cells express significant levels of 12- lipoxygenase.
  • FIGS 2, 2A and 2B are drawings showing partial 12-LOX cDNA and deduced amino acid sequence alignment.
  • E2 exon 2; 12: intron 2; bp: base pair; Pl,
  • PCR products were electrophoretically separated in a 2% agarose gel. The sequences are unique. Fragments at the predicted size from human (H) platelet and Clone A, rat (R) platelet and W256, mouse (M) platelet and B16a cells, were purified and sequenced. 12-LOX cDNA sequence and deduced amino acid sequence were aligned by the Jotun-Hein method.
  • Figures 2C and 2D are a drawing and a chart showing phylogenetic tree and sequence distances, respectively, based on the limited sequences obtained from PCR fragments. H 12-LOX, R 12-LOX and M
  • 12-LOX human, rat and mouse 12-LOX; Pro: protein.
  • Sequences derived from Clone A, W256 and B16a are identical to that of human, rat and mouse platelet, respectively.
  • Figures 3A and 3B are graphs showing 12-HETE production in tumor cells.
  • Particulate fractions of cultured W256 ( Figure 3A) and B16a cells ( Figure 3B) were incubated with 9.2 ⁇ M of 1 C-arachidonic acid for 15 minutes at 37°C.
  • AA metabolites were analyzed by reverse phase (RP) high pressure liquid chromatography (HPLC) .
  • RP reverse phase
  • HPLC high pressure liquid chromatography
  • Figure 4 is a photomicrograph showing in situ hybridization for 12-LOX in normal prostate tissue. Positive staining (arrow) is restricted to basal cells, with no staining present in normal secretory cells.
  • Figures 5A and 5B are photomicrographs showing in situ hybridization for 12-LOX in a focus of prostatic adenocarcinoma. Reactivity with sense probe is demonstrated on the left panel (Figure 5A) and with antisense probe in the right panel ( Figure 5B) . Only the antisense probe causes staining.
  • Figure 6 is a photomicrograph showing in situ hybridization of 12-LOX in an area of high grade prostatic intraepithelial neoplasia (PIN) . There is variable staining pattern with intense cytoplasmic reactivity in the secretory cells. In one of the involved glands only a focal area of the high grade PIN shows positive staining (arrow) .
  • Figure 7 is a photomicrograph showing in situ hybridization for 12-LOX demonstrating intense positive reactivity in the cytoplasm of a focus of moderately differentiated adenocarcinoma (PCA) and high grade prostatic intraepithelial neoplasia (PIN) . There is negative reactivity in the secretory cells of the uninvolved normal prostatic epithelium (N, double arrows) .
  • PCA moderately differentiated adenocarcinoma
  • PIN prostatic intraepithelial neoplasia
  • Figure 8 is a photomicrograph showing in situ hybridization for 12-LOX demonstrating strong positive cytoplasmic reactivity in a focus of moderately differentiated (Gleason score 6) prostatic adenocarcinoma (PCA) .
  • the adjacent normal glands (N) show only focal reactivity of the basal cells.
  • Figures 9A and 9B are photomicrographs showing in situ hybridization for 12-LOX in a case of moderately differentiated (Gleason score 7) prostatic adenocarcinoma (PCA) .
  • the photomicrograph from the left panel ( Figure 9A) shows an area of non-reactivity in the center of the tumor and the right panel ( Figure 9B) shows an area of moderate reactivity at the edge of the neoplasm.
  • Figure 10 is a photomicrograph showing in situ hybridization of 12-LOX in a poorly differentiated prostatic adenocarcinoma (Gleason score 9) showing positive reactivity within the tumor cell cytoplasm.
  • the adjacent normal epithelium (N) shows complete absence of reactivity.
  • Figure 11 is a photomicrograph showing a case of prostatic adenocarcinoma studied by in situ hybridization for 12-LOX. Note the moderate intense reactivity within the tumor cells within the prostate gland (PCA) . The tumor nests (M) which have invaded beyond the prostatic capsule (C) show intense cytoplasmic staining.
  • Figure 12 is a gel showing the immunoreactivity of the anti-12-LOX peptide antibody toward platelet 12-LOX protein.
  • Figure 14 is a schematic view of a plasmid including a segment of 12 LOX.
  • Figure 15 shows the DNA of the segment of Figure 14 (SEQ ID NO: 14 and SEQ ID NO: 15).
  • Figure 16 is a southern blot showing identification of platelet-type 12-LOX in human prostate cancer by RT-PCR. N is normal and T is tumor. C is control. DESCRIPTION OF PREFERRED EMBODIMENTS
  • the present invention relates to a method for detecting an RNA for 12-lipoxygenase from cells which comprises: extracting the RNA from the cells; binding the RNA with spaced apart sense and antisense primers defining a region of the DNA of the 12-lipoxygenase which is unique; and reverse transcribing the RNA bound with the sense and the antisense primers with a polymerase to provide a cDNA from the RNA; and detecting the RNA of the 12-lipoxygenase from the cDNA.
  • the present invention relates to a DNA substantially free of other DNA comprising a sequence sufficiently duplicative of that set forth in the group consisting of SEQ ID NOS: 5, 6, 7, 8 and 9 such that the
  • DNA selectively hybridizes under stringent conditions to
  • the present invention also relates to a DNA which is a segment of 12-lipoxygenase and selected from the group consisting of SEQ ID NOS. 5, 6, 7, 8 and 9
  • the present invention relates to a peptide free of other peptides and allelic or species variants thereof which is a segment of 12-lipoxygenase and derived from a DNA substantially free of other DNA comprising a sequence sufficiently duplicative of that set forth in the group consisting of SEQ ID NOS: 5, 6, 7, 8 and 9 such that the DNA selectively hybridizes under stringent conditions to DNA encoding 12-LOX. Further, the present invention relates to a peptide which is a segment of human 12-lipoxygenase and selected from the group consisting of SEQ ID NOS. 5, 6, 7, 8, and 9 (Appendix I) substantially free from other peptides.
  • the present invention relates to a method for detecting 12-lipoxygenase or a 12- lipoxygenase RNA which comprises: binding the 12- lipoxygenase or RNA with a labeled DNA substantially free of other DNA comprising a sequence sufficiently duplicative of that set forth in the group consisting of SEQ ID NOS: 5, 6, 7, 8 and 9 such that the DNA selectively hybridizes under stringent conditions to DNA encoding 12-LOX.
  • the present invention relates to a method for detecting 12-lipoxygenase or a 12- lipoxygenase RNA which comprises: binding the 12- lipoxygenase or RNA with a labeled DNA selected from the group consisting of SEQ ID NOS: 5, 6, 7, 8 and 9 (Appendix I) and detecting the DNA bound 12-lipoxygenase or RNA.
  • the present invention also relates to a DNA primer selected from the group consisting of SEQ ID NOS: 1, 2, 3 and 4 (Appendix I) substantially free of other DNA.
  • the present invention relates to a method for detecting 12-lipoxygenase which comprises reacting a Western Blot containing cellular 12-lipoxygenase with an antibody to the 12-lipoxygenase derived from SEQ ID NO: 8 (Appendix I) to form a complex of the 12-lipoxygenase and the antibody, and detecting the complex.
  • the present invention relates to a method for detecting an RNA of 12-lipoxygenase by reverse transcribing the RNA and detecting a first cDNA produced by the reverse transcribing, the improvement which comprises: providing as an internal standard with the RNA a segment of the RNA which is reversed transcribed and detected as a second cDNA along with the first cDNA.
  • oligo probes amino acid and nucleotide sequences of human 5-, 12- and 15-lipoxygenase were aligned by Jotun-Hein method with weight table. Five regions (RI, RII, RIII, RIV and RV) with least homology among these three enzymes were identified. Oligo primers derived from these five regions are specific to human 12-LOX enzyme. They differ 30-70% in sequence compared to human 5- and 15- LOX sequences and any other sequences in the GenBank (Intell Genetics, Inc., Mountainview, CA) . If primer sequence shifts one codon to upstream or downstream, specificity will decrease 5-10%. In addition, peptide sequence RIV is used to generate anti-peptide polyclonal antibodies. The sequences are shown in Table 1 as follows:
  • RII a.a.116-123 CCA GGA GAC AAT GCT TTG GAC ATG Pro Gly Asp Asn Ala Leu Asp Met
  • RIII a.a.151-158 ACC ATC GCT GCA GAC CGT AAG GAT Thr lie Ala Ala Asp Arg Lys Asp
  • RV a.a.455-462 CTC CCA GGT GCT CTC TAT GCC CAT Leu Pro Gly Ala Leu Tyr Ala His
  • the present invention particularly relates to a method for detecting an RNA for 12-lipoxygenase from cells which comprises: extracting the RNA from the cells; binding the RNA with a sense primer selected from the group consisting of 5'-CCTTCCCGTGCTACCGCTGG-3 ' and 5'-CAGGAGACAATGCTTTGGAC-3 ' as set forth in sequence ID NOS. 1 and 2 and an antisense primer selected from the group consisting of: 5-TGGGGTTGGCACCATTGAGG-3 ⁇ and
  • amino acid residues are: EDTA, ethylenediamine tetraacetate; FCS, fetal calf serum; FITC, fluorescein isothiocyanate conjugated; HBSS, Hank's balanced salt solution; MEM, minimum essential medium; phosphate buffered saline; PMA, phorbol 12- myristate-13-acetate, RT-PCR, reverse transcription- polymerase chain reaction; RP-HPLC, reverse phase high performance liquid chromatography; AA, arachidonic acid.
  • amino acid residues are: EDTA, ethylenediamine tetraacetate; FCS, fetal calf serum; FITC, fluorescein isothiocyanate conjugated; HBSS, Hank's balanced salt solution; MEM, minimum essential medium; phosphate buffered saline; PMA, phorbol 12- myristate-13-acetate, RT-PCR, reverse transcription- polymerase chain reaction; RP-HPLC, reverse phase
  • the cDNA derived from the polymerase chain reaction can be detected by conventional methods such as DNA-DNA probes, binding the cDNA with a labeled oligoprobe which conforms to the cDNA; by the use of labeled monoclonal or polyclonal antibodies which selectively bind to the cDNA and by labeling the cDNA which is detected.
  • the label can be, for instance, an enzyme, radiolabel or various chemiluminescent labels.
  • the enzyme is reacted with a substrate to produce a detectable product such as light or color. This method can be as described in U.S. Patent No. 5,004,565 to Schaap, for instance.
  • Labeled nucleotides can be used in the PCR. All of these methods are well known to those skilled in the art.
  • the cDNA is used as a probe to detect the DNA or 12-lipoxygenase directly and can be labeled.
  • Example 1 The expression of 12-lipoxygenase in tumor cells was particularly determined by RT-PCR and sequencing. The 12(S)-HETE production was analyzed by HPLC.
  • mRNAs from human, rat and mouse platelets as well as human colon carcinoma (Clone A) , rat Walker carcinoma (W256) and mouse melanoma (B16a) were reverse transcribed and amplified by polymerase chain reaction with platelet 12- lipoxygenase specific primers. Identity of the PCR fragments was confirmed by sequencing. Tumor cell- derived 12-HETE was determined by HPLC analysis. MATERIALS AND METHODS
  • Tumor cell lines Human colon carcinoma (Clone A) , rat
  • Walker carcinoma (W256) , mouse melanoma (B16a) and Lewis lung carcinoma (3LL) were cultured in MEM medium supplemented with 5% FCS.
  • RNA extraction Total RNA from different tumor cells as well as platelets was obtained using Guanidine thiocyanate-CsCl method (Chang, Y.S., et al., Int. J. Cancer 51, 445-451 (1992a)). Total RNA from cells was suspended at l ⁇ g/ ⁇ l in water. RNA from platelets was suspended in a total volume of 10 ⁇ l.
  • RT- PCR Reverse transcription-polvmerase chain reaction
  • PCT primers were designed according to the human platelet 12-lipoxygenase cDNA sequence (Izu i, T., et al., Proc. Natl. Acad. Sci. USA 87,7477-7481 (1990)) and were capable of amplifying 12-LOX sequences from mouse and rat platelets.
  • First run PCR profile was 94°C, 30 sec; 60°C, 30 sec and 72°C, 1 min for 35 cycles with sense (S'-CCTTCCCGTGCTACCGCTGG- 3') and anti-sense (5 '-TGGGGTTGGCACCATTGAGG-3 • ) primers.
  • Nested PCR profile was 94°C, 30 sec; 50°C, 30 sec and 72°C, 1 min for 35 cycles with nested primers (sense 5'- CAGGAGACAATGCTTTGGAC-3 • , anti-sense 5 ' - GAACAACTCATCATCCTGCC-3 ' ) . These are set forth in SEQ ID NOS: 1 to 4.
  • 12-lipoxy ⁇ enase mRNA in tumor cells Expression 12-lipoxy ⁇ enase mRNA in tumor cells. Expression of 12-lipoxygenase was examined in human colon carcinoma (Clone A) , rat Walker carcinoma (W256) , mouse Lewis lung carcinoma (3LL) and amelanotic melanoma (B16a) cells. Total RNA was isolated from each cell line, reverse transcribed to cDNA and amplified by the polymerase chain reaction. Human, rat and mouse platelet RNAs were used as positive controls. Fragments at the predicted size (338 bp) were obtained from human, rat and mouse platelets ( Figure 1) . This result indicates that PCR primers designed according to the human platelet 12-LOX cDNA sequence are suitable for both rat and mouse platelet 12-LOX sequences.
  • 12(S)-HETE production in tumor cells. Although 12-LOX mRNA was identified in tumor cells, it is also important to demonstrate the presence of 12-LOX enzyme or its metabolite, i.e., 12(S)-HETE. 12 (S) -HETE production was determined in W256 and B16a cells. Cultured tumor cells were lysed and subfractionated. The particulate and cytosolic fractions were incubated for 15 min with R e ⁇ labeled arachidonic acid (9.2 ⁇ M) and arachidonic acid metabolites were analyzed by RP-HPLC.
  • tumor cell 12-lipoxygenase was studied at mRNA, metabolite and function levels; (2) it is suggested that tumor cells respond not only to exogenous 12 (S)-HETE generated by other cells but also to endogenous 12(S)-HETE.
  • the exogenous and tumor cell- derived endogenous 12(S)-HETEs can act in synergy during tumor cell-platelet-endothelium.
  • the 12-lipoxygenase (12-LOX) sequence was partially determined from human, rat and mouse platelets.
  • Human 12-LOX has approximately 92% homology with mouse 12-LOX and about 90% with rat 12-LOX within the PCR amplified region, whereas mouse 12-LOX shares about 97% similarity with rat 12-LOX.
  • Human, rat and mouse 12-LOX seem to share high homology.
  • Bovine 12-LOX has approximately 88% similarity to porcine 12-LOX. However, it appears that there is only about 40% homology comparing bovine and porcine 12-LOX to human, rat and mouse 12-LOX.
  • tumor cell 12- LOX is responsible for the generation of endogenous 12 (S)-HETE.
  • 12 (S)-HETE was not always produced by cultured cells. It was observed 12 (S) -HETE production in 3 out of 5 experiments using W256 carcinoma cells and in 3 out of 10 experiments using B16a melanoma cells. However, when these cultured cells were injected (subcutaneously) into animals, corresponding solid tumors removed and monodispersed tumor cells isolated by centrifugal elutriation, 12 (S)-HETE production was consistently observed. The reason for this discrepancy is currently unknown. It is possible that certain factors required for 12-LOX activation are not always present under culture conditions. The 12-LOX activation could be at two levels, i.e., transcriptional and post- transcriptional levels.
  • the human 12-LOX promoter contains putative GC box (4 sites) , CACCC box (2 sites), AP-2 binding motif (3 sites) and glucocorticoid-responsive element (GRE, 1 site) (Yoshimoto, T., et al., Proc. Natl. Acad. Sci. USA 87, 2124-2146 (1990)) .
  • the GRE by its definition, is activated by glucocorticoid, however, also by androgen, mineralocorticoids and progesterone (Faisst and Meyer, Nucleic Acids Res. 20:3-26 (1992)).
  • the AP-2 is inducible by TPA, cAMP and retinoic acid (Faisst and Meyer, Nucleic Acids Res. 20:3-26 (1992)) . Indeed, it has been shown that treatment of HEL cells with 160 nM TPA for 24 hours increases 12-LOX mRNA (Funk, C. D. , et al., J. Biol. Chem. 266, 12508-12513 (1991)) and 12-LOX enzyme activity (Izumi, T. , et al., Proc. Natl. Acad. Sci. USA 87, 7477-7481 (1990)).
  • Example 2 This example shows human testing for prostatic carcinoma.
  • RNA Isolation Total RNA from tissues was isolated by guanidinium-isothiocyanate/CsCl gradient centrifugation method with modifications. Briefly, tissue sample up to 0.5g was homogenized in Tempest IQ
  • CsCl cesium chloride
  • IM EDTA IM EDTA
  • sequences of the primers SEQ ID NOS: 1 to 4 used were: 5 '-CCT TCC CGT GCT ACC GCT GG-3" (12LOX, sense), 5'-TGG GGT TGG CAC CAT TGA GG-3' (12LOX antisense), 5'-CAG GAG ACA ATG CTT TGG AC-3' (12LOX, nested sense), 5 '-GAA CAA CTC ATC ATC ATC CTG CC-3 • (12LOX nested antisense), 5 '-ACT GGC GTC TTC ACC ACC AT-3 ' (GAPDH, sense), and 5 *-TGA CCT TGC CCA CAG CCT TG-3 ' (GAPDH, antisense) (glyceraldehydes-3- phosphate dihydrogenase SEQ ID NOS: 10 and 11) .
  • RNA Reverse transcription One ⁇ g of total RNA was reverse transcribed in 20 ⁇ l solution containing 50 mM Tris pH8.3, 75 mM KCl, 3 mM MgCl 2 , 5 mM DTT, 0.5 mM dNTP, 75 ng/ml of BSA, 30 units of RNasin (Promega, Madison, WI) , 2 ⁇ M of anti-sense primer, 200 units of MMLV reverse transcriptase (Gibco BRL) at room temperature for 10 min and 37°C for 1 hour.
  • RNasin Promega, Madison, WI
  • Polymerase chain reaction One ⁇ l of reverse transcription mixture was amplified by PCR in 100 ⁇ l buffer containing 10 mM Tris pH 8.3, 50 mM KCl, 2.5-4 mM MgCl 2 , 0.1 mg/ml BSA, 1 ⁇ M of sense and anti-sense primers and 2.5 unit AmpliTaq DNA polymerase (Perkin Elmer Cetus, Newark, CT) .
  • the PCR was run in the Gene Amp PCR system 9600 (Perkin Elmer Cetus) at 94°C, 30 sec; 49-58°, 30 sec and 72°C, 1 min for 25-30 cycles.
  • RNA from different tissues was amplified by RT-PCR for glyceraldehyde-3- phosphate dehydrogenase (GAPDH) (Tso et al., Nucleic
  • DNA sequencing The Sanger dideoxynucleotide- mediated termination method was used with the Sequenase Version 2.0 DNA sequencing kit (USB, Cleveland, OH). Plasmid sequencing was done according to the manufacturer's instructions. For direct sequencing of PCR products, the following modifications were done. Agarose gel purified double strand cDNA fragment and primer were denatured at 100°C for 5 min and annealed on dry ice for 2. min. The DNA chain was labeled using [ 35 S]-dATP and extended on ice for 10 min with Sequenase, and terminated at 45°C for 10 min. The samples were loaded onto 6% DNA sequencing gel (BaseAce with temperature controller, Stratagene, LaJolla, CA) .
  • the same sample was loaded twice at a 1 hr interval, migrated for a total of 2 hrs at 45°C and at an average of 100 watts.
  • the gel was fixed, dried and exposed to x-ray film.
  • DNA sequencing was performed on both strands to confirm the accuracy.
  • the data was recorded by a sequence gel reader (IBI, New Haven, CT) which was connected to MacVECTOR (IBI) on a Macintosh Ilci computer.
  • Gel Densitometry Scanning The intensity of individual bands was measured by densitometry scanning using an LKB 2400 GelScan XL laser densitometer (Pharmacia, Piscataway, NJ) .
  • mRNA expression of 12-LOX was calculated by normalization of the amount of the RT-PCR amplified fragments to the 367 bp GAPDH fragment.
  • Table 2 shows the results of testing for increased mRNA in numerous patients.
  • Prostate tissue was obtained from prostate glands removed at the time of radical prostatectomy for clinically localized prostatic adenocarcinoma. All tissues had been fixed in 10% neutral buffered formalin and embedded in paraffin. Representative sections were selected which included areas of normal and hyperplastic prostatic tissue, prostatic intraepithelial neoplasia, and prostatic adenocarcinoma. Sections were cut at 5 microns and placed on charged glass slides (PROBEON PLUS microscope slides, Fischer Scientific, Pittsburgh, PA) .
  • Oligonucleotides cDNA probes of Beta 3, a subunit of integrins receptors family, and 12- lipoxygenase (12LOX) an enzyme involved in the metabolism of arachidonic acid, were used using the Mac VECTOR 4.0 sequence analysis software package (IBI, New Haven, CT) .
  • the 12-LOX antisense probe was 20 base oligonucleotide of sequence 5 » -CGC-GGT-TGT-ACG-ACC-CGG- AGU-3' (SEQ ID NO: 12).
  • the sequence of the 12-LOX sense probe was 5 • -CTC-CGG-GTC-GTA-CAA-CCG-CGU-3 » (SEQ ID NO: 13) .
  • the cDNA probes were synthesized by Research Genetics, Huntsville, AL.
  • the lyophilized oligonucleotide probes were reconstituted to a stock concentration of l ⁇ g/ ⁇ l with 10 mM Tris base, 1 mM EDTA buffer pH 7.5. The working concentration was determined following titration of the probes.
  • the stock solution of beta 3 was diluted 1:2000, while the stock solution of 12-LOX was diluted 1:1000 with Probe Diluent (Research Genetics) just before use.
  • ISH experiment was carried out using the Microprobe System staining station (Fisher Scientific, Houston, TX) , following Fidler's protocol with few modifications.
  • the samples were preheated for 20 minutes at 60°C, mounted on the slide holder, dewaxed for 5 minutes with a mixture of 3 parts of limolene (HEMO-DE; Fisher Scientific, Houston, TX) and 1 part of xylene followed by xylene for 4 minutes, 100% and 95% ethanol for 1 minute each.
  • the slides were then treated with pepsin
  • Tris buffer 0.1 M Tris-Cl pH 7.5; 0.1 M NaCl; 5 mM MgCl 2 ; 0.25% (v/v) of 30% brij 35
  • acetylated 0.25% acetic anhydride in 0.1 M triethanolamine
  • the slides were incubated with a monoclonal anti-biotin antibody at a concentration of 2 to 4 ⁇ g/ml (Boehringer Mannheim, Indianapolis, IN) for 90 minutes at 37°C in order to block non-specific reaction with endogenous biotin.
  • the antibody was purchased from Boehringer as lyophilisat, dissolved in 1 ml of DEPC treated H 2 0 obtaining a final concentration of 100 ⁇ /ml then aliquoted in anti-biotin wash buffer (40 mM Tris- HC1 pH 7.4; 150 mM NaCl; 1% (v/v) Tween 20) .
  • the slides were then washed with anti-biotin wash buffer 6x1 minute at ambient temperature.
  • test slides were hybridized with the appropriate probe.
  • the positive control sample was hybridized with a poly dT cDNA probe while the negative control was hybridized with the probe diluent only.
  • concentration of the probes was 1 ⁇ g/ml.
  • the hybridization was carried out for 40 minutes at 52°C starting at a temperature of 105°C, in order to anneal the RNAs, and decreasing the temperature gradually until it reached 52°C. The decline of the temperature from 105°C to 52°C took about 4 minutes.
  • 2xSSC-SDS-Brij 2xSSC (sodium chloride, sodium citrate); 1% (v/v) SDS (sodium dodecyl sulfonate) ; 0.25% (v/v) 30% H 2 0 Brij (detergent)) 4x3 min, 3x3 minutes with O.lxSSC-SDS-Brij (O.lxSSC, 1%(v/v) SDS; 0.25% (v/v) 30% H 2 0 Brij) .
  • 2xSSC-SDS-Brij 2xSSC-SDS-Brij
  • Example 4 Generation of anti-12-LOX specific antisera.
  • IgG was purified from the antisera using a combination of ammonia sulfate precipitation and Protein A resin. Briefly, one volume of serum was mixed with an equal volume of 0.15M NaCl. To the mixture, ammonium sulfate (313 g/1) was then added. After 2 hours of stirring, precipitated proteins were obtained by centrifugation (10,000 x g; 10 min), resuspended in a small volume of 0.15M NaCl, and dialyzed against phosphate buffered saline (PBS) . Afterwards, the sample was mixed with one volume of loading buffer (1.5M Glycine, 3M NaCl, pH 8.9) and loaded onto a 5-ml Protein A column.
  • loading buffer 1.5M Glycine, 3M NaCl, pH 8.9
  • the column was washed with 20 ml of loading buffer and IgG was eluted with 100 mM citric acid, pH 4. The eluate was neutralized with 1 M Tris-HCl, pH 7.4 and dialyzed against PBS. The IgG preparation was then aliquoted and stored at -20°C.
  • Platelets of human, rat or mouse were prepared as previously described (Steinert, et al., Int. J. Cancer 54:92-101 (1993)) . Briefly, blood was mixed with one volume of sodium citrate anticoagulant and centrifuged at 50 x g for 30 minutes. The supernatant was saved. The pellet was mixed with 1/3 volume of platelet wash (Ca 2+ , Mg 2+ and phenol red free MEM) containing 0.8% of 0.1M EDTA and then centrifuged at 50 x g for 20 minutes. The supernatant was saved and the pellet was washed two more times. The supernatants were pooled and centrifuged at 700 x g for 10 minutes.
  • the pellet (washed platelets) was resuspended in phosphate buffered saline to approximately 10 8 / ⁇ l.
  • platelets were sonicated (4 x 15 sec) in PBS containing 0.5 ⁇ M PMSF and 1 ⁇ g/ml leupeptin. The sonicate was then centrifuged at 100,000 x g for 60 minutes at 4°C and the supernatant was saved. Protein content was measured by the Bradford dye binding assay using bovine serum albumin as standard.
  • Non-specific binding sites on the membrane was blocked with BLOTTO (10% non-fat dry milk in PBS containing 0.05% TWEEN-20, Sigma Chemical, St. Louis, MO) .
  • BLOTTO 50% non-fat dry milk in PBS containing 0.05% TWEEN-20, Sigma Chemical, St. Louis, MO
  • the membrane was then incubated for 1.5 hours with anti 12-LOX IgG diluted 1:1000 in BLOTTO followed by incubation for 1 hour with Horse radish peroxidase conjugated goat anti-rabbit antibody (1:5000 in BLOTTO) . Following extensive wash with PBS containing 0.05% TWEEN-20, the bound secondary antibody was revealed by the ECL Western blot detection system (Amersham Life Science, Little Chalfort, Buckinghamshire, England) . The results were recorded on the Amersham HYPERFILM-ECL.
  • 12-LOX antibody (1:1000 in PBS) was preincubated for 60 minutes with human platelet cytosol or bovine serum albumin (50 ⁇ g/ml) before incubation with blocked nitrocellulose membrane.
  • the anti-12-LOX peptide antibody detected a single band of approximately 72 kD in the purified human/HEL 12-LOX sample. It picked up a strong single band in the cytosol from human and mouse platelets, and a much weaker band in cytosol of rat platelets. The molecular weight of the bands in platelet cytosol samples was slightly smaller than that in the purified 12-LOX.
  • Example 5 This example shows an internal control for quantification of 12-LOX mRNA by RT-PCR.
  • RT-PCT is a sensitive and rapid method to detect gene expression.
  • RT-PCR results can be affected by conditions for synthesis of DNA molecules from a RNA preparation, effectiveness of DNA molecule amplification during PCR cycles and the richness of target molecules in experimental samples. Therefore, including an internal control during RT-PCR eliminates the variations between experiments as well as subtle differences among individual reaction tubes.
  • FIG. 14 and 15 An internal control for the RT-PCR of 12-LOX gene was developed as shown in Figures 14 and 15.
  • a fragment of DNA was cloned into plasmid.
  • the DNA fragment comprises 12-LOX specific sequences at the left and right ends (represented by arrows) .
  • the 12-LOX sequence fragment is flanked by two promoters (signal necessary for synthesis of RNA from DNA) .
  • the construct is introduced into E. coli bacterium and thus replicated to large quantities.
  • the construct is used by synthesis of 12-LOX sequence RNA.
  • a known amount of the 12-LOX sequence RNA is included in each RT-PCR reaction as internal control. Signal derived from the 12-LOX control RNA is easily differentiated from signal derived from 12-LOX gene RNA.
  • 12-LOX gene RNA signal is normalized to 12-LOX control RNA signal. Therefore, quantification of 12-LOX is achieved by referencing the same internal control.
  • the construct is a plasmid with 12-LOX sequence fragment.
  • the plasmid is pBlueScript SK (Stratagene, LaJolla, CA) .
  • the promoters are SP6 and T7.
  • the fragment sequence is shown in
  • Figure 15 shows the presence of enhanced levels of platelet 12-LOX in prostate cancer tissue.
  • One ⁇ g of total RNA from matching normal prostate tissue samples and prostate cancer tissue samples from the same patient were used to synthesize cDNA and amplified by PCR as in Example 2.
  • Equal volume of PCR products (15 ⁇ l) was separated on a 1.0% agarose gel and transferred, crosslinked, hybridized with a radioactive p 32 probe prepared from platelet-type 12 '-LOX cDNA fragment.
  • the membrane was washed under high stringency conditions and exposed to x-ray film at -80°C. Result from 5 pairs of sample is shown.
  • N normal tissue
  • T tumor tissue.
  • the results clearly show the enhanced amounts of 12-LOX in the prostate cancer tissue.
  • Table 3 shows the results in tabular form.
  • T2 confined to gland (non-invasive); T3: extension beyond gland (invasive); NO: no lymph ⁇ node metastases; Nl: lymph node metastases present; B: African American; C: Caucasian. ' Positive: more than 50% increase of 12-LOX expression in cancer tissue compared to matching normal tissue.
  • TNM staging is "Tumor Lymph node metastasis”.
  • Example 7 Detection of elevated 12-lipoxygenase expression -in human prostatic carcinoma with Cytokeratin correction.
  • 12-lipoxygenase (12-LOX) mRNA levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) from matching tumor and normal tissues from same patients. Same initial total RNA loading for cDNA synthesis was controlled by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Tso et al. Nucleic Acids Research, .13, 2485-2502 (1985)) which is ubiquitously expressed in all cell types including both epithelial and stromal cells.
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • Cytokeratin 18 (Leube et al., Differentiation 33:69-85 (1986); Oshi a et al., Differentiation 33:61-68 (1986)) served as an epithelial maker for this purpose for the following reasons: i) .
  • Cytokeratins including cytokeratin 18 have been shown to be expressed exclusively in cells of epithelial origin (Hallowes et al., Anticancer Research 11: 1799-1806 (1991); Vachon et al., The Anatomical Record 236:567-576 (1993); ii) .
  • Immunochemistry studies using antibodies against cytokeratin 18 showed that normal epithelial cells and cancerous epithelial (carcinoma) cells expressed same levels of cytokeratin 18. Therefore, mRNA expression of 12-LOX by normalization of the amount of the RT-PCR amplified fragments to the GAPDH fragment was calculated and then the value corrected to the cytokeratin 18 expression level.
  • the exclusion criterion for elevated expression of 12-LOX mRNA used in Table 4 is a value larger than 25% increase in 12-LOX from cancer tissue compared to matching normal tissue from same patient.
  • a larger than 50% elevation of 12-LOX was used in tumor tissue verses normal tissue as a cutoff for consideration of positive elevation. With the refinement of the assay (i.e., cytokeratin correction), this value was lowered to >25% elevation.
  • Tissue specimens Matching prostatic carcinoma and normal tissues were obtained from 50 patients undergone radical prostatectomy. Tissue samples were immediately frozen at -80°C. A 3- ⁇ m section was cut from each tissue and stained with hematoxylin/eosin. Samples containing no carcinoma cells were considered normal and samples containing more than 70% carcinoma cells were considered tumor.
  • Total RNA Isolation Total RNA from tissues was isolated by guanidinium-isothiocyanate/CsCl gradient centrifugation method with modifications.
  • tissue sample up to 0.5g was homogenized in Tempest IQ Homogenizer) in 3.5 ml of guanidinium isothiocyanate (GT) solution containing 4M GT, IM NaAc (pH 5.0), and 0.5M DTT. Then the lysate was transferred into a Ti 55 tube containing 1.3 ml of cesium chloride (CsCl) solution (5.7M CsCl and 0.1M EDTA (pH 6.5)). Centrifuged in Ti 55 rotor (Beckman XL-90 Ultracentrifuge) at 42,000 rpm, 20°C for at least 12 hours. The pellet was suspended in 300 ⁇ l of cold 10 mM Tris (pH 8.0). The sample was then phenol- extracted, ethanol-precipitated, and resuspended in distilled water. The optical density was then measured at 260 nm (wavelength) in Pharmacia Ultrospec III Spectrophotometer.
  • GT guani
  • Oligonucleatides synthesis Oligonucleatides designed for PCR were synthesized in the Gene Assembler Plus (Pharmarcia LKB) . The sequences of the primers used were: 5'-CCT TCC CGT GCT ACC GCT GG-3' (12LOX, sense), 5'-TGG GGT TGG CAC CAT TGA GG-3 1 (12-LOX antisense), 5'-CAG GAG ACA ATG CTT TGG AC-3' (12-LOX, nested sense) , 5'-GAA CAA CTC ATC ATC ATC ATC CTG CC-3 • (12-LOX nested antisense), 5'-ACT GGC GTC TTC ACC ACC AT-3 1 (GAPDH, sense), and 5'-TGA CCT TGC CCA CAG CCT TG-3• (GAPDH, antisense), 5•-GAA GAA GAA CCA CGA AGA GGA AG-3' (cytokeratin 18, sense, SEQ ID NO: 16),
  • RNA Reverse transcription One ⁇ g of total RNA was reverse transcribed in 20 ⁇ l solution containing 50 mM Tris pH 8.3, 75 mM KCl, 3 mM MgCl 2 , 5 mM DTT, 0.5 mM dNTP, 75 ng/ml of BSA, 30 units of RNasin (Promega) , 2 ⁇ M of anti-sense primer, 200 units of MMLV reverse transcriptase (Gibco BRL) at room temperature for 10 min and 37°C for 1 h.
  • RNasin Promega
  • Polymerase chain reaction One ⁇ l of reverse transcription mixture was amplified by PCR in 100 ⁇ l buffer containing 10 mM Tris pH 8.3, 50 mM KCl, 2.5 mM MgCl 2 , 0.1 mg/ml BSA, 1 ⁇ M of sense and anti-sense primers and 2.5 unit AmpliTaq DNA polymerase (Perkin Elmer Cetus) .
  • the PCR was run in the GeneAmp PCR system 9600 (Perkin Elmer Cetus) at 94°C, 30 sec; 49- 58°C, 30 sec and 72°C, l min for 25-30 cycles.
  • 1 ⁇ l of first PCR product was reamplified under the same conditions but with nested primers.
  • RNA from different tissues was amplified by RT-PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Tso et al., Nucleic Acids Research, Volume 13, Number 7, 2485-2502 (1985)) and cytokeratin 18 (Leube et al. , Differentiation 33:69-85 (1986); Oshima et al. , Differentiation 33:61-68 (1986) expression using GAPDH and cytokeratin 18 specific primers in separate tubes.
  • GAPDH reaction is used to confirm that equal amounts of total RNA were reverse transcribed (Maier et al., Science, Vol. 249, 1570-1574 (1990); Maier et al.
  • cytokeratin 18 reaction serves as a correction factor for epithelial cell content.
  • the conditions of RT-PCR for GAPDH and cytokeratin 18 were the same as that of 12-LOX.
  • One tenth (10 ⁇ l) of PCR products were electrophoresed on 2% agarose gels, which were then visualized by ethidium bromide staining and photographed.
  • DNA sequencing The Sanger dideoxynucleotide-mediated termination method was used with the Sequenase Version 2.0 DNA sequencing kit (USB, Cleveland, OH) . Plasmid sequencing was done according to the manufacturer's instructions. For direct sequencing of PCR products, the following modifications were done. Agarose gel purified double strand cDNA fragment and primer were denatured at 100°C for 5 minutes and annealed on dry ice for 2 minutes. The DNA chain was labeled using [ 35 S]-dATP and extended on ice for 10 minutes with Sequenase, and terminated at 45°C for 10 minutes. The samples were loaded onto 6% DNA sequencing gel (Base Ace with temperature controller, Stratagene) .
  • the same sample was loaded twice at a 1 hour interval, migrated for a total of 2 hours at 45°C and at an average of 100 watts.
  • the gel was fixed, dried and exposed to x-ray film.
  • DNA sequencing was performed on both strands to confirm the accuracy.
  • the data was recorded by a sequence gel reader (IBI, New Haven, CT) which was connected to MacVECTOR (IBI, New Haven, CT) on a Macintosh Ilci computer.
  • mRNA expression of 12-LOX was calculated by normalization of the amount of the RT-PCR amplified fragments to the 367 bp GAPDH fragment, then corrected by the cytokeratin 18 expression level.
  • T2 confined to gland (non-invasive); T3: extension beyond gland (invasive); NO: no lymph ⁇ node metastases; Nl: lymph node metastases present; B: African American; C: Caucasian. Positive: more than 25% increase of 12-LOX expression in cancer tissue compared to matching normal tissue.

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Abstract

Methods for detecting 12-lipoxygenase or the DNA or RNA encoding the 12-lipoxygenase involving DNA, RNA or antibody detection. The method is particularly used to detect metastatic tumor cells which produce 12-lipoxygenase in vitro as an indicator of disease.

Description

METHODS FOR DETECTING 12-LIPOXYGENASE AND ITS ENCODING DNA
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to methods for detecting 12-lipoxygenase or the DNA encoding this enzyme. In particular the present invention relates to a method using antibody or DNA-DNA probe binding for the detection. The present invention also relates to the use of a polymerase chain reaction to produce a cDNA encoding a unique part of 12-lipoxygenase which is detected.
(2) Description of Related Art
Metastasis is a cascade which requires multiple factors expressed by tumor cells as well as surrounding stromal cells, and is subject to distinct stimulus regulations (Hart, I. R. , et al., Biocim. Biophys. Acta. 989:65-84, (1989)) . During the metastatic process, tumor cells undergo extensive cell- cell and cell-matrix interactions. Two cell types, i.e., platelets and endothelial cells with which tumor cells interact during hematogenous metastasis are capable of producing a vast array of lipid mediators by either direct or transcellular metabolism of precursors (Chen, Y. Q., et al., Cancer Metast. Rev. 11, 389-410 (1992a)). Platelets are capable of converting arachidonic acid (AA) to prostaglandins and predominantly to thromboxane A2 through the cyclooxygenase (COX) pathway. Platelets also metabolize AA through the lipoxygenase (LOX) pathway producing 12 (S) -hydroxyeicosatetraenoic acid (12(S)-HETE) (Moncada, S., and Higgs, E. A., Clin. Heamato. 15: 273- 292 (1986)). After intravasation, tumor cells interact with platelets and induce platelet aggregation with concomitant synthesis of platelet 12(S)-HETE (Honn, K. V., et al., Biochem. Biophys. Res. Co mun. 29:384-389 (1987); Steinert, B. W. , et al., Int. J. Cancer (54:92- 101 (1993); Honn, K. V., et al., Exptl. Cell Res. 210: 1-9, (1994)). Therefore, platelets can be a contributor of 12(S)-HETE to facilitate tumor cell interaction with endothelial cells and subendothelial matrix. Previously, it has been shown that exogenous 12(S)-HETE increases tumor cell adhesion to endothelium (Honn, K. V., et al., Biochem. Biophys. Res. Commun. 29:384-389 (1987) ; Grossi, I. M. , et al., Cancer Res. 49:1029-1037 (1989)) , and induces large vessel and microvessel endothelial cell retraction (Honn, K. V., et al., FASEB J. 3:2285-2293 (1989); Tang, D. , et al., Kluwer Academic Publishers, pp219-229 (1992) ; Honn, K. V., et al., Cancer Res. 54:565-574 (1994)) and stimulates tumor cell spreading on matrix (Ti ar, J. , et al., Int. J. Cancer 52, 594-603 (1992)). Tumor cells express a variety of adhesion molecules. Among them, integin αιrbjS3 is an important receptor for tumor cell-host cell and tumor cell-matrix interaction (Chen, Y. Q. , et al., J. Biol. Chem. 267, 17314-17320 (1992b); Chang, . C. , et al., Int. J. Cancer 51, 445-451, (1992a)). Pretreatment of tumor cells with exogenous 12(S)-HETE enhances their adhesion to and spreading on fibronectin, which is mediated exclusively by <^^3 receptors and in a cytoskeleton-dependent manner (Chopra, H., et al., Int. J. Cancer 49:774-786 (1991) ; Timar, J. , et al., Int. J. Cancer 52, 594-603 (1992)) . Besides the effects on integrins, 12(S)-HETE also appears to regulate other factors which are important to tumor cell metastasis. Pretreatment of tumor cells with 12(S)-HETE stimulates cell motility (Raz, A., et al., Kluwer Academic Publishers, pp645-649 (1992) ; Timar, et al, Intl. J. Cancer 55:1003-1010 (1993)) and release of cathepsin B (Sloane, B. F. , et al., Kluwer Academic Publishers, pp373-377 (1991)). Interestingly, it was observed that treatment of 256 cells with 12(S)-HETE induced a 100% ■ increase in membrane-associated protein kinase C (PKC) activity (Liu, B. , et al., C. Cell Regulation 2,1045- 1055 (1991)). This activation of PKC by 12(S)-HETE was also observed in B16a cells. Since only the PKCα isozyme was found in B16a cells by western blotting, it suggests that 12(S)-HETE can up-regulate PKCα activity in B16a cells.
Based on these effects of exogenous 12(S)-HETE on tumor cells and endothelium, it was believed that during the tumor cell-platelet-endothelium interaction, tumor cells induce platelet aggregation and, in response, platelets release 12(S)-HETE. On one hand, the platelet-derived 12(S)-HETE induces endothelial cell retraction and exposure of subendothelial matrix. On the other hand, 12(S)-HETE activates tumor cell protein kinase C which in turn triggers cytoskeleton rearrangement and thus increases integrin αIIb/33 surface expression, consequently enhances tumor cell adhesion to and spreading on endothelium. In addition, 12(S)-HETE increases tumor cell motility and release of protease.
Therefore, 12(S)-HETE augments the rate of tumor cell extravasation and finally metastasis. Thus, 12(S)-HETE has significant importance in hematogenous metastasis.
There was a need for a reliable assay method determining the presence of and level of tumor cell 12-lipoxygenase which produces the 12(S)-HETE.
OBJECTS
It is therefore an object of the present invention to provide a method for detecting 12- lipoxygenase. Further, it is an object of the present invention to provide a method for detecting 12- lipoxygenase in tumor cells. It is further an object of the present invention to provide a method which is relatively simple, reliable and economical. These and other objects will become increasingly apparent by reference to the following description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an electrophoresis gel showing 12- lipoxygenase expression in various types of tumor cells. Total RNA from each cell line was reverse transcribed and amplified by polymerase chain reaction (RT-PCR) . Ten μl of the final PCR products were separated on 2% agarose gel. Control RT-PCR was run under the same conditions without total RNA. λEcoRV:λ DNA EcoRV fragment as marker; H Platelet: human platelet; R Platelet: rat platelet; M Platelet: mouse platelet. Tumor cells express significant levels of 12- lipoxygenase.
Figures 2, 2A and 2B are drawings showing partial 12-LOX cDNA and deduced amino acid sequence alignment. E2: exon 2; 12: intron 2; bp: base pair; Pl,
P2, P3, P4: primer 1, 2, 3 and 4 respectively. PCR products were electrophoretically separated in a 2% agarose gel. The sequences are unique. Fragments at the predicted size from human (H) platelet and Clone A, rat (R) platelet and W256, mouse (M) platelet and B16a cells, were purified and sequenced. 12-LOX cDNA sequence and deduced amino acid sequence were aligned by the Jotun-Hein method. Figures 2C and 2D are a drawing and a chart showing phylogenetic tree and sequence distances, respectively, based on the limited sequences obtained from PCR fragments. H 12-LOX, R 12-LOX and M
12-LOX: human, rat and mouse 12-LOX; Pro: protein.
Sequences derived from Clone A, W256 and B16a are identical to that of human, rat and mouse platelet, respectively.
Figures 3A and 3B are graphs showing 12-HETE production in tumor cells. Particulate fractions of cultured W256 (Figure 3A) and B16a cells (Figure 3B) were incubated with 9.2 μM of 1C-arachidonic acid for 15 minutes at 37°C. AA metabolites were analyzed by reverse phase (RP) high pressure liquid chromatography (HPLC) . In the experiment presented here, approximately 1% of added AA was converted to 12-HETE (3030 cpm) in W256 cells and 0.85% (3078 cpm) in B16a cells. Total radioactivity recovery was greater than 95%. Five independent experiments were performed with W256 cells and comparable results were obtained in 3 out of 5 experiments. Ten independent experiments were performed with B16a cells and comparable results were obtained in 3 out of 10 experiments.
Figure 4 is a photomicrograph showing in situ hybridization for 12-LOX in normal prostate tissue. Positive staining (arrow) is restricted to basal cells, with no staining present in normal secretory cells.
Figures 5A and 5B are photomicrographs showing in situ hybridization for 12-LOX in a focus of prostatic adenocarcinoma. Reactivity with sense probe is demonstrated on the left panel (Figure 5A) and with antisense probe in the right panel (Figure 5B) . Only the antisense probe causes staining.
Figure 6 is a photomicrograph showing in situ hybridization of 12-LOX in an area of high grade prostatic intraepithelial neoplasia (PIN) . There is variable staining pattern with intense cytoplasmic reactivity in the secretory cells. In one of the involved glands only a focal area of the high grade PIN shows positive staining (arrow) .
Figure 7 is a photomicrograph showing in situ hybridization for 12-LOX demonstrating intense positive reactivity in the cytoplasm of a focus of moderately differentiated adenocarcinoma (PCA) and high grade prostatic intraepithelial neoplasia (PIN) . There is negative reactivity in the secretory cells of the uninvolved normal prostatic epithelium (N, double arrows) .
Figure 8 is a photomicrograph showing in situ hybridization for 12-LOX demonstrating strong positive cytoplasmic reactivity in a focus of moderately differentiated (Gleason score 6) prostatic adenocarcinoma (PCA) . The adjacent normal glands (N) show only focal reactivity of the basal cells.
Figures 9A and 9B are photomicrographs showing in situ hybridization for 12-LOX in a case of moderately differentiated (Gleason score 7) prostatic adenocarcinoma (PCA) . The photomicrograph from the left panel (Figure 9A) shows an area of non-reactivity in the center of the tumor and the right panel (Figure 9B) shows an area of moderate reactivity at the edge of the neoplasm.
Figure 10 is a photomicrograph showing in situ hybridization of 12-LOX in a poorly differentiated prostatic adenocarcinoma (Gleason score 9) showing positive reactivity within the tumor cell cytoplasm. The adjacent normal epithelium (N) shows complete absence of reactivity.
Figure 11 is a photomicrograph showing a case of prostatic adenocarcinoma studied by in situ hybridization for 12-LOX. Note the moderate intense reactivity within the tumor cells within the prostate gland (PCA) . The tumor nests (M) which have invaded beyond the prostatic capsule (C) show intense cytoplasmic staining.
Figure 12 is a gel showing the immunoreactivity of the anti-12-LOX peptide antibody toward platelet 12-LOX protein.
Human, rat, or mouse platelet cytosol (25 μg each) or 12-LOX protein (10 ng) was separated on a 10% SDS-PAGE gel and transferred to nitrocellulose membrane. 12-LOX in the samples were blotted and incubated with IgG purified from 8-week bleed of anti-12-LOX peptide antisera and revealed with the ECL reagents. The molecular standards used are from a Sigma Chemicals (St. Louis, MO) SDS-7B kit. Figures 13A, 13B and 13C are gels showing specificity of the anti-12-LOX peptide antibody. Prior to incubation with the nitrocellulose membrane, anti-12- LOX peptide antibody (1:1000 in PBS) was preincubated for 60 minutes with human platelet cytosol or BSA (50 μg/ml) .
Figure 14 is a schematic view of a plasmid including a segment of 12 LOX.
Figure 15 shows the DNA of the segment of Figure 14 (SEQ ID NO: 14 and SEQ ID NO: 15).
Figure 16 is a southern blot showing identification of platelet-type 12-LOX in human prostate cancer by RT-PCR. N is normal and T is tumor. C is control. DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention relates to a method for detecting an RNA for 12-lipoxygenase from cells which comprises: extracting the RNA from the cells; binding the RNA with spaced apart sense and antisense primers defining a region of the DNA of the 12-lipoxygenase which is unique; and reverse transcribing the RNA bound with the sense and the antisense primers with a polymerase to provide a cDNA from the RNA; and detecting the RNA of the 12-lipoxygenase from the cDNA.
The present invention relates to a DNA substantially free of other DNA comprising a sequence sufficiently duplicative of that set forth in the group consisting of SEQ ID NOS: 5, 6, 7, 8 and 9 such that the
DNA selectively hybridizes under stringent conditions to
DNA encoding 12-LOX. Preferably there is at least 80% homology. The present invention also relates to a DNA which is a segment of 12-lipoxygenase and selected from the group consisting of SEQ ID NOS. 5, 6, 7, 8 and 9
(Appendix I) substantially free from other segments.
The present invention relates to a peptide free of other peptides and allelic or species variants thereof which is a segment of 12-lipoxygenase and derived from a DNA substantially free of other DNA comprising a sequence sufficiently duplicative of that set forth in the group consisting of SEQ ID NOS: 5, 6, 7, 8 and 9 such that the DNA selectively hybridizes under stringent conditions to DNA encoding 12-LOX. Further, the present invention relates to a peptide which is a segment of human 12-lipoxygenase and selected from the group consisting of SEQ ID NOS. 5, 6, 7, 8, and 9 (Appendix I) substantially free from other peptides.
Further, the present invention relates to a method for detecting 12-lipoxygenase or a 12- lipoxygenase RNA which comprises: binding the 12- lipoxygenase or RNA with a labeled DNA substantially free of other DNA comprising a sequence sufficiently duplicative of that set forth in the group consisting of SEQ ID NOS: 5, 6, 7, 8 and 9 such that the DNA selectively hybridizes under stringent conditions to DNA encoding 12-LOX.
Further still, the present invention relates to a method for detecting 12-lipoxygenase or a 12- lipoxygenase RNA which comprises: binding the 12- lipoxygenase or RNA with a labeled DNA selected from the group consisting of SEQ ID NOS: 5, 6, 7, 8 and 9 (Appendix I) and detecting the DNA bound 12-lipoxygenase or RNA.
The present invention also relates to a DNA primer selected from the group consisting of SEQ ID NOS: 1, 2, 3 and 4 (Appendix I) substantially free of other DNA.
The present invention relates to a method for detecting 12-lipoxygenase which comprises reacting a Western Blot containing cellular 12-lipoxygenase with an antibody to the 12-lipoxygenase derived from SEQ ID NO: 8 (Appendix I) to form a complex of the 12-lipoxygenase and the antibody, and detecting the complex.
Finally, the present invention relates to a method for detecting an RNA of 12-lipoxygenase by reverse transcribing the RNA and detecting a first cDNA produced by the reverse transcribing, the improvement which comprises: providing as an internal standard with the RNA a segment of the RNA which is reversed transcribed and detected as a second cDNA along with the first cDNA.
In order to design specific oligo probes, amino acid and nucleotide sequences of human 5-, 12- and 15-lipoxygenase were aligned by Jotun-Hein method with weight table. Five regions (RI, RII, RIII, RIV and RV) with least homology among these three enzymes were identified. Oligo primers derived from these five regions are specific to human 12-LOX enzyme. They differ 30-70% in sequence compared to human 5- and 15- LOX sequences and any other sequences in the GenBank (Intell Genetics, Inc., Mountainview, CA) . If primer sequence shifts one codon to upstream or downstream, specificity will decrease 5-10%. In addition, peptide sequence RIV is used to generate anti-peptide polyclonal antibodies. The sequences are shown in Table 1 as follows:
TABLE 1
12-Lipoχyqenase
SEQ Region Nucleotide Sequence encoding ID NO: Amino Acid
RI: a.a.16-21 C TCC GGG TCG TAC AAC CGC G Ser Gly Ser Tyr Asn Arg
RII: a.a.116-123 CCA GGA GAC AAT GCT TTG GAC ATG Pro Gly Asp Asn Ala Leu Asp Met
RIII: a.a.151-158 ACC ATC GCT GCA GAC CGT AAG GAT Thr lie Ala Ala Asp Arg Lys Asp
RIV a.a.176-193 TGG ACA CTG AAG GCA GGG GCT polyclonal Trp Thr Leu Lys Ala Gly Ala antibody CTG GAG ATG GCC CTC AAA CGT Leu Glu Met Ala Leu Lys Arg GTT TAC ACC CTC CTG Val Tyr Thr Leu
RV a.a.455-462 CTC CCA GGT GCT CTC TAT GCC CAT Leu Pro Gly Ala Leu Tyr Ala His
The present invention particularly relates to a method for detecting an RNA for 12-lipoxygenase from cells which comprises: extracting the RNA from the cells; binding the RNA with a sense primer selected from the group consisting of 5'-CCTTCCCGTGCTACCGCTGG-3 ' and 5'-CAGGAGACAATGCTTTGGAC-3 ' as set forth in sequence ID NOS. 1 and 2 and an antisense primer selected from the group consisting of: 5-TGGGGTTGGCACCATTGAGG-3 ■ and
5-GAACAACTCATCATCCTGCC-3 • as set forth in SEQ ID NOS. 3 and 4; reverse transcribing the RNA bound with the sense and the antisense primers with a polymerase to provide a cDNA from the RNA; and detecting the RNA .for the 12- lipoxygenase from the cDNA.
The abbreviations used in the present invention are: amino acid residues are: EDTA, ethylenediamine tetraacetate; FCS, fetal calf serum; FITC, fluorescein isothiocyanate conjugated; HBSS, Hank's balanced salt solution; MEM, minimum essential medium; phosphate buffered saline; PMA, phorbol 12- myristate-13-acetate, RT-PCR, reverse transcription- polymerase chain reaction; RP-HPLC, reverse phase high performance liquid chromatography; AA, arachidonic acid. The cDNA derived from the polymerase chain reaction (PCR) can be detected by conventional methods such as DNA-DNA probes, binding the cDNA with a labeled oligoprobe which conforms to the cDNA; by the use of labeled monoclonal or polyclonal antibodies which selectively bind to the cDNA and by labeling the cDNA which is detected. The label can be, for instance, an enzyme, radiolabel or various chemiluminescent labels. The enzyme is reacted with a substrate to produce a detectable product such as light or color. This method can be as described in U.S. Patent No. 5,004,565 to Schaap, for instance. Labeled nucleotides can be used in the PCR. All of these methods are well known to those skilled in the art. The cDNA is used as a probe to detect the DNA or 12-lipoxygenase directly and can be labeled.
Example 1 The expression of 12-lipoxygenase in tumor cells was particularly determined by RT-PCR and sequencing. The 12(S)-HETE production was analyzed by HPLC.
In the present invention, mRNAs from human, rat and mouse platelets as well as human colon carcinoma (Clone A) , rat Walker carcinoma (W256) and mouse melanoma (B16a) were reverse transcribed and amplified by polymerase chain reaction with platelet 12- lipoxygenase specific primers. Identity of the PCR fragments was confirmed by sequencing. Tumor cell- derived 12-HETE was determined by HPLC analysis. MATERIALS AND METHODS
Tumor cell lines. Human colon carcinoma (Clone A) , rat
Walker carcinoma (W256) , mouse melanoma (B16a) and Lewis lung carcinoma (3LL) were cultured in MEM medium supplemented with 5% FCS.
Platelets. Ten ml whole blood from human, rat or mouse were drawn into 2 ml of citrate solution (3.8% sodium citrate, 4.8% glucose, 0.9% sodium chloride, pH 7.5) . Samples were centrifuged at 200x g for 15 min at room temperature. The supernatant was recovered, and the pellet was washed with 1 ml of HBSS and recentrifuged. Supernatants were combined and spun at 800x g for 10 min. The pellet (i.e. platelets) was then used for RNA extraction. RNA extraction. Total RNA from different tumor cells as well as platelets was obtained using Guanidine thiocyanate-CsCl method (Chang, Y.S., et al., Int. J. Cancer 51, 445-451 (1992a)). Total RNA from cells was suspended at lμg/μl in water. RNA from platelets was suspended in a total volume of 10 μl.
Reverse transcription-polvmerase chain reaction (RT- PCR) . One μg of total RNA from each cell line or 1 μl of RNA from platelets were reverse transcribed using gene specific primers under conditions described previously (Chen, Y. Q. , et al 1992). One μl of reverse transcription mixture was amplified by PCR in 100 μl buffer (Chen et al., 1992). DNase (Gibco, BRL) were used to prevent both PCR product carry-over and genomic DNA contamination. The PCR was run in the GENEAMP PCR system 9600 (Perkin Elmer Cetus) . PCT primers were designed according to the human platelet 12-lipoxygenase cDNA sequence (Izu i, T., et al., Proc. Natl. Acad. Sci. USA 87,7477-7481 (1990)) and were capable of amplifying 12-LOX sequences from mouse and rat platelets. First run PCR profile was 94°C, 30 sec; 60°C, 30 sec and 72°C, 1 min for 35 cycles with sense (S'-CCTTCCCGTGCTACCGCTGG- 3') and anti-sense (5 '-TGGGGTTGGCACCATTGAGG-3 • ) primers. Nested PCR profile was 94°C, 30 sec; 50°C, 30 sec and 72°C, 1 min for 35 cycles with nested primers (sense 5'- CAGGAGACAATGCTTTGGAC-3 • , anti-sense 5 ' - GAACAACTCATCATCCTGCC-3 ' ) . These are set forth in SEQ ID NOS: 1 to 4.
DNA sequencing. PCR products were separated and sequenced as described previously (Chen, Y. Q. , J. Biol. Chem. 267, 17314-17320 (1992b)) . Data was analyzed using the LASERGENE DNASTAR software (Madison, WI) on a Macintosh Ilci computer (Cupertina, LA) .
HPLC Analysis. Cultured W256 and B16a cells were washed
(3x) with ice-cold PBS and then scraped in 1 ml of 0.1
M HEPES (pH 8) per 75 cm2 flask. Cells were sonicated
(3x10 sec) on ice. The lysate was centrifuged at 100,000 g for 60 min at 4°C. The resulting supernatant was designated as the cytosol fraction and the pellet as particulate fraction. The particulate fraction was resuspended in HEPES and protein concentration was determined by the Bradford method (Bradford, M. M. , Anal. Biochem. 72, 248-254 (1976)) using BSA as standard. An aliquot containing 2 mg protein was incubated for 15 min at 37°C with 0.5 μCi (9.22 μM) of
14C-AA in 1 ml of 0.1 M HEPES (pH 8) . The reaction was terminated by' acidification to pH 3.5 with 1 N HC1 and lipids were extracted (3x) with 3 volumes of hexane- ethyl acetate (1:1). Organic layers were pooled, dried and analyzed by RP-HPLC (reverse phase high pressure liquid chromatography) as described previously (Liu, B. , et al., C. Cell Regulation 2,1045-1055 (1991)) . Adhesion. Fibronectin- or subendothelial matrix-coated plates were prepared (Chang, Y. S., et al., Int. J.
Cancer 51, 445-451 (1992a)) and adhesion assays were performed following established protocols (Grossi, I.
M., et al., Cancer Res. 49: 1029-1037 (1989)). RESULTS
Expression 12-lipoxyαenase mRNA in tumor cells. Expression of 12-lipoxygenase was examined in human colon carcinoma (Clone A) , rat Walker carcinoma (W256) , mouse Lewis lung carcinoma (3LL) and amelanotic melanoma (B16a) cells. Total RNA was isolated from each cell line, reverse transcribed to cDNA and amplified by the polymerase chain reaction. Human, rat and mouse platelet RNAs were used as positive controls. Fragments at the predicted size (338 bp) were obtained from human, rat and mouse platelets (Figure 1) . This result indicates that PCR primers designed according to the human platelet 12-LOX cDNA sequence are suitable for both rat and mouse platelet 12-LOX sequences. Fragments at identical size were also obtained from Clone A, W256, 3LL and B16a cells, although the intensity was varied (Clone A» B16a > W256 K 3LL) (Fig. 1) . In order to prevent carry-over contamination, RNA samples and all other reagents except primer oligos were treated with DNase and negative controls were included in every RT- PCR experiment. In order to prevent genomic DNA contamination, primers were targeted at different exons (E2, E3 and E6) . Therefore, the fragment amplified from RNA should be 338 bp whereas the fragment from genomic DNA should be 1045 bp (Figure 2) .
The 338 bp fragments from human, rat and mouse platelets as well as human Clone A, rat W256 and mouse B16a cells were purified and sequenced. Approximately 200 bp sequence information were obtained from each fragment by direct sequencing PCR products. Sequences of human Clone A, rat W256 and mouse B16a matched 100% to those of human, rat and mouse platelets, respectively. Using the limited sequence information, 12-LOX cDNAs and deduced amino acid sequences were aligned by the Jotun-Hein method and the sequence distances were calculated based on the amino acid sequence (Figures 2A and 2B) . Human 12-LOX has approximately 92% homology. with mouse 12-LOX and about 90% with rat '12-LOX within the PCR amplified region, whereas mouse 12-LOX shares about 97% similarity with rat 12-LOX.
12(S)-HETE production in tumor cells. Although 12-LOX mRNA was identified in tumor cells, it is also important to demonstrate the presence of 12-LOX enzyme or its metabolite, i.e., 12(S)-HETE. 12 (S) -HETE production was determined in W256 and B16a cells. Cultured tumor cells were lysed and subfractionated. The particulate and cytosolic fractions were incubated for 15 min with Re¬ labeled arachidonic acid (9.2 μM) and arachidonic acid metabolites were analyzed by RP-HPLC. Two major metabolites which coeluted with authentic 15 (S) -HETE and 12 (S)-HETE were detectable in both cell lines with the particulate fraction (Figures 3A and 3B) . No 12 (S) -HETE production was observed with cytosolic fraction. In the present invention, it was demonstrated tumor cells are capable of producing 12 (S) -HETE, and like exogenous 12 (S)-HETE the endogenously synthesized 12-HETE can play a role in tumor cell metastasis. This finding is important in several aspects: (1) For the first time, tumor cell 12-lipoxygenase was studied at mRNA, metabolite and function levels; (2) it is suggested that tumor cells respond not only to exogenous 12 (S)-HETE generated by other cells but also to endogenous 12(S)-HETE. The exogenous and tumor cell- derived endogenous 12(S)-HETEs can act in synergy during tumor cell-platelet-endothelium.
By RT-PCR and sequencing, the 12-lipoxygenase (12-LOX) sequence was partially determined from human, rat and mouse platelets. Human 12-LOX has approximately 92% homology with mouse 12-LOX and about 90% with rat 12-LOX within the PCR amplified region, whereas mouse 12-LOX shares about 97% similarity with rat 12-LOX. Human, rat and mouse 12-LOX seem to share high homology. Bovine 12-LOX has approximately 88% similarity to porcine 12-LOX. However, it appears that there is only about 40% homology comparing bovine and porcine 12-LOX to human, rat and mouse 12-LOX. Recently, it has been proposed that human 12-LOX was functionally differentiated by a gene duplication from a common ancestor LOX for 12- and 15-lipoxygenases at certain stage of evolution, and later the porcine 12-LOX was differentiated from 15-LOXs (Toh, H. , et al., Prostaglandins 44, 291-315 (1992)) . cDNA encoding 12- LOX has been cloned from human (Funk, C. D., et al., Proc. Natl. Acad. Sci. USA 87, 5638-5642 (1990) ; Izumi, T. , et al., Proc. Natl. Acad. Sci. USA 87, 7477-7481 (1990); and DeMarzo, N. , et al., Am. J. Physiol. 262, L198-L207 (1992)) strongly suggest that tumor cell 12- LOX is responsible for the generation of endogenous 12 (S)-HETE.
12 (S)-HETE was not always produced by cultured cells. It was observed 12 (S) -HETE production in 3 out of 5 experiments using W256 carcinoma cells and in 3 out of 10 experiments using B16a melanoma cells. However, when these cultured cells were injected (subcutaneously) into animals, corresponding solid tumors removed and monodispersed tumor cells isolated by centrifugal elutriation, 12 (S)-HETE production was consistently observed. The reason for this discrepancy is currently unknown. It is possible that certain factors required for 12-LOX activation are not always present under culture conditions. The 12-LOX activation could be at two levels, i.e., transcriptional and post- transcriptional levels. First, the human 12-LOX promoter contains putative GC box (4 sites) , CACCC box (2 sites), AP-2 binding motif (3 sites) and glucocorticoid-responsive element (GRE, 1 site) (Yoshimoto, T., et al., Proc. Natl. Acad. Sci. USA 87, 2124-2146 (1990)) . The GRE, by its definition, is activated by glucocorticoid, however, also by androgen, mineralocorticoids and progesterone (Faisst and Meyer, Nucleic Acids Res. 20:3-26 (1992)). The AP-2 is inducible by TPA, cAMP and retinoic acid (Faisst and Meyer, Nucleic Acids Res. 20:3-26 (1992)) . Indeed, it has been shown that treatment of HEL cells with 160 nM TPA for 24 hours increases 12-LOX mRNA (Funk, C. D. , et al., J. Biol. Chem. 266, 12508-12513 (1991)) and 12-LOX enzyme activity (Izumi, T. , et al., Proc. Natl. Acad. Sci. USA 87, 7477-7481 (1990)). Secondly, it has been reported that there is a 12-LOX specific inhibitory factor in cytosol of the human epidermoid carcinoma A431 cells (Chang, W. C, et al., J. Biol. Chem. 267, 3657- 3666 (1992b)). This factor inhibits cytosolic 12-LOX but not the membrane-associated 12-LOX activity. A similar inhibitory factor was also found in the cytosol fraction of cultured epithelial cells (Shornick, L. P. , et al., J. Biol. Chem. 268, 371-376 (1993)) . Therefore, it is plausible that tumor cell 12-LOX activity can be regulated by translocation. 12-HETE production was observed with particulate fraction but not with cytosolic fraction.
Example 2 This example shows human testing for prostatic carcinoma.
MATERIALS AND METHODS
Tissue specimens. Matching prostatic carcinoma and normal tissues were obtained from 50 patients who had undergone radical prostatectomy Tissue samples were immediately frozen at -80°C. A 3-μm section was cut from each tissue and stained with hematoxylin/eosin. Samples containing no carcinoma cells were considered normal and samples containing more than 70% carcinoma cells were considered tumor. Total RNA Isolation. Total RNA from tissues was isolated by guanidinium-isothiocyanate/CsCl gradient centrifugation method with modifications. Briefly, tissue sample up to 0.5g was homogenized in Tempest IQ
Homogenizer) in 3.5 ml of guanidinium isothiocyanate (GT) solution containing 4M GT, IM NaAc (pH 5.0), and
0.5M DTT. Then the lysate was transferred into a Ti 55 tube containing 1.3 ml of cesium chloride (CsCl) solution (5.7M CsCl and 0. IM EDTA (pH 6.5)) . Centrifuged in Ti 55 rotor (Beckman XL-90 Ultracentrifuge, Palo Alto, CA) at 42,000 rpm, 20°C for at least 12 hours. Suspended the pellet in 300 μl of cold 10 mM Tris (pH 8.0). The sample was then phenol- extracted, ethanol-precipitated, and resuspended in distilled water. The optical density was then measured at 260 nm (wavelength) in Pharmacia ULTROSPEC III Spectrophotometer (Piscataway, NJ) . Oligonucleotides synthesis: Oligonucleotides designed for PCT were synthesized in the Gene Assembler Plus (Pharmacia LKB, Piscataway, NJ) . The sequences of the primers SEQ ID NOS: 1 to 4 used were: 5 '-CCT TCC CGT GCT ACC GCT GG-3" (12LOX, sense), 5'-TGG GGT TGG CAC CAT TGA GG-3' (12LOX antisense), 5'-CAG GAG ACA ATG CTT TGG AC-3' (12LOX, nested sense), 5 '-GAA CAA CTC ATC ATC ATC ATC CTG CC-3 • (12LOX nested antisense), 5 '-ACT GGC GTC TTC ACC ACC AT-3 ' (GAPDH, sense), and 5 *-TGA CCT TGC CCA CAG CCT TG-3 ' (GAPDH, antisense) (glyceraldehydes-3- phosphate dihydrogenase SEQ ID NOS: 10 and 11) .
Reverse transcription: One μg of total RNA was reverse transcribed in 20 μl solution containing 50 mM Tris pH8.3, 75 mM KCl, 3 mM MgCl2, 5 mM DTT, 0.5 mM dNTP, 75 ng/ml of BSA, 30 units of RNasin (Promega, Madison, WI) , 2 μM of anti-sense primer, 200 units of MMLV reverse transcriptase (Gibco BRL) at room temperature for 10 min and 37°C for 1 hour.
Polymerase chain reaction: One μl of reverse transcription mixture was amplified by PCR in 100 μl buffer containing 10 mM Tris pH 8.3, 50 mM KCl, 2.5-4 mM MgCl2, 0.1 mg/ml BSA, 1 μM of sense and anti-sense primers and 2.5 unit AmpliTaq DNA polymerase (Perkin Elmer Cetus, Newark, CT) . The PCR was run in the Gene Amp PCR system 9600 (Perkin Elmer Cetus) at 94°C, 30 sec; 49-58°, 30 sec and 72°C, 1 min for 25-30 cycles. For nested PCR, 0.1-1 μl of first PCR product was reamplified under the same conditions but with nested primers. Same amount of total RNA from different tissues was amplified by RT-PCR for glyceraldehyde-3- phosphate dehydrogenase (GAPDH) (Tso et al., Nucleic
Acids Research, J13_, 2485-2502 (1985)) expression using GAPDH specific primers in separate tubes to confirm that equal amounts of total RNA were reverse transcribed
(Maier et al., J. of Biol. Chem. 265. 10805-10808 (199) ;
Maier et al, Science 249, 1570-1574 (1990)). The conditions of RT-PCR for GAPDH were the same as that of 12-LOX. One tenth (10 μl) of PCR products were electrophoresed on 2% agarose gels, which were then visualized by ethidium bromide staining and photographed.
DNA sequencing: The Sanger dideoxynucleotide- mediated termination method was used with the Sequenase Version 2.0 DNA sequencing kit (USB, Cleveland, OH). Plasmid sequencing was done according to the manufacturer's instructions. For direct sequencing of PCR products, the following modifications were done. Agarose gel purified double strand cDNA fragment and primer were denatured at 100°C for 5 min and annealed on dry ice for 2. min. The DNA chain was labeled using [35S]-dATP and extended on ice for 10 min with Sequenase, and terminated at 45°C for 10 min. The samples were loaded onto 6% DNA sequencing gel (BaseAce with temperature controller, Stratagene, LaJolla, CA) . The same sample was loaded twice at a 1 hr interval, migrated for a total of 2 hrs at 45°C and at an average of 100 watts. The gel was fixed, dried and exposed to x-ray film. DNA sequencing was performed on both strands to confirm the accuracy. The data was recorded by a sequence gel reader (IBI, New Haven, CT) which was connected to MacVECTOR (IBI) on a Macintosh Ilci computer. Gel Densitometry Scanning. The intensity of individual bands was measured by densitometry scanning using an LKB 2400 GelScan XL laser densitometer (Pharmacia, Piscataway, NJ) .
Data Analysis. mRNA expression of 12-LOX was calculated by normalization of the amount of the RT-PCR amplified fragments to the 367 bp GAPDH fragment. RESULTS
Table 2 shows the results of testing for increased mRNA in numerous patients.
Table 2
12LOX (increase over normal tissue)
T2 3/12(25%)
T3 15/20(75%)
LGS 12/23(52%)
HGS 6/9(66%) NO 16/27(59%)
Nl 2/5(40%)
Patient samples were run for 12-lipoxygenase by RT-PCR on fifty (50) patients. Table 2 shows the percentage of patients in each subgroup which had elevated 12-LOX message (mRNA) levels when their tumor tissue was compared to a piece of normal tissue run in a similar fashion from the same patient. These are matched control versus tumor tissue. An elevated level of 50% above normal tissue was set as the arbitrary cut- off point. This may be too high an elevation, however, it will take further refinement of the data to be able to determine whether this figure can be lowered. A larger number of stage T3 (invasive carcinoma) patients had higher 12-LOX message level than stage T2 (low invasive) patients. A correlation thus far with Gleason score (LGS and HGS) was not found. A low Gleason score
(less than 6) indicates a more favorable prognosis than a high Gleason score. Lymph node negative patients (NO) versus node positive patients (Nl) do not show a clear trend based upon limited data.
Example 3
The technique for in situ hybridization as in Figures 4 to 11 is described. MATERIALS AND METHODS Human prostate tissue sections
Prostate tissue was obtained from prostate glands removed at the time of radical prostatectomy for clinically localized prostatic adenocarcinoma. All tissues had been fixed in 10% neutral buffered formalin and embedded in paraffin. Representative sections were selected which included areas of normal and hyperplastic prostatic tissue, prostatic intraepithelial neoplasia, and prostatic adenocarcinoma. Sections were cut at 5 microns and placed on charged glass slides (PROBEON PLUS microscope slides, Fischer Scientific, Pittsburgh, PA) .
In situ hybridization results were assessed by two observers. Intensity of reaction was evaluated in stroma, normal epithelium, premalignant epithelium
(prostatic intraepithelial neoplasia) and malignant epithelium (prostatic adenocarcinoma) . In all cases the intensity of staining between control and test slides was compared. Oligonucleotide Probes
Oligonucleotides cDNA probes of Beta 3, a subunit of integrins receptors family, and 12- lipoxygenase (12LOX) , an enzyme involved in the metabolism of arachidonic acid, were used using the Mac VECTOR 4.0 sequence analysis software package (IBI, New Haven, CT) . The 12-LOX antisense probe was 20 base oligonucleotide of sequence 5 »-CGC-GGT-TGT-ACG-ACC-CGG- AGU-3' (SEQ ID NO: 12). The sequence of the 12-LOX sense probe was 5 •-CTC-CGG-GTC-GTA-CAA-CCG-CGU-3 » (SEQ ID NO: 13) . All the probes have 6 molecules of biotin incorporated at the Brigati tail. The cDNA probes were synthesized by Research Genetics, Huntsville, AL. The lyophilized oligonucleotide probes were reconstituted to a stock concentration of lμg/μl with 10 mM Tris base, 1 mM EDTA buffer pH 7.5. The working concentration was determined following titration of the probes. The stock solution of beta 3 was diluted 1:2000, while the stock solution of 12-LOX was diluted 1:1000 with Probe Diluent (Research Genetics) just before use.
In Situ Hybridization flSH) . ISH experiment was carried out using the Microprobe System staining station (Fisher Scientific, Houston, TX) , following Fidler's protocol with few modifications.
The samples were preheated for 20 minutes at 60°C, mounted on the slide holder, dewaxed for 5 minutes with a mixture of 3 parts of limolene (HEMO-DE; Fisher Scientific, Houston, TX) and 1 part of xylene followed by xylene for 4 minutes, 100% and 95% ethanol for 1 minute each. The slides were then treated with pepsin
(Boehringer Mannheim, Indianapolis, IN) 2μg/ml in 0.1 N
HC1 for 4 minutes at 105°C, rinsed 3x30 sec with Tris buffer (0.1 M Tris-Cl pH 7.5; 0.1 M NaCl; 5 mM MgCl2; 0.25% (v/v) of 30% brij 35) , acetylated (0.25% acetic anhydride in 0.1 M triethanolamine) for 10 minutes, then washed with Tris buffer pH 7.5 for 3x1 minutes.
The slides were incubated with a monoclonal anti-biotin antibody at a concentration of 2 to 4 μg/ml (Boehringer Mannheim, Indianapolis, IN) for 90 minutes at 37°C in order to block non-specific reaction with endogenous biotin. The antibody was purchased from Boehringer as lyophilisat, dissolved in 1 ml of DEPC treated H20 obtaining a final concentration of 100 μ/ml then aliquoted in anti-biotin wash buffer (40 mM Tris- HC1 pH 7.4; 150 mM NaCl; 1% (v/v) Tween 20) . The slides were then washed with anti-biotin wash buffer 6x1 minute at ambient temperature. The test slides were hybridized with the appropriate probe. The positive control sample was hybridized with a poly dT cDNA probe while the negative control was hybridized with the probe diluent only. For 12-LOX the concentration of the probes was 1 μg/ml. The hybridization was carried out for 40 minutes at 52°C starting at a temperature of 105°C, in order to anneal the RNAs, and decreasing the temperature gradually until it reached 52°C. The decline of the temperature from 105°C to 52°C took about 4 minutes.
After hybridization, the samples were washed with 2xSSC-SDS-Brij (2xSSC (sodium chloride, sodium citrate); 1% (v/v) SDS (sodium dodecyl sulfonate) ; 0.25% (v/v) 30% H20 Brij (detergent)) 4x3 min, 3x3 minutes with O.lxSSC-SDS-Brij (O.lxSSC, 1%(v/v) SDS; 0.25% (v/v) 30% H20 Brij) . 1% BSA was used 3x1 minutes to block protein- protein interactions followed by incubation with avidin- alkaline phosphatase ((Boehringer) 1:2000) in 1% BSA for 30 minutes. A brief rinse of 30 sec with lx avidin- alkaline phosphatase enhancer (Biomeda, Foster City, CA) was applied to the samples before incubation with fresh chromogene substrate; McGadey reagent (0.67% (v/v) NBT; •0.33% (v/v) BCIP in 1 ml Tris buffer pH 9.5) for 20 minutes at 40°C. The samples were washed 3x1 minutes with H20 Brij, counterstained with Fast Red 2x1 minutes and finally washed for 1 minute with H20 Brij, covered with Crystal Mount, heat dried, and mounted with Permount. Results
The results are shown in Figures 4 to 11. As can be seen the prostatic cells stained at the leading edge of the tumor.
Example 4 1. Generation of anti-12-LOX specific antisera.
The synthesis of LOX peptides and the generation of antibodies were conducted as a custom order by Research Genetics (Huntville, AL) . 12-LOX specific peptides were synthesized using the Fmoc solid phase methods utilizing MAP resin technology (Tam, J. P., Proc. Natl. Aca. Sci., USA 85, 5409-5413 (1988)) . Rabbits were immunized with the peptides and bled 4, 8 and 10 weeks post immunization. The sequence used is set forth in SEQ ID NO: 8.
2. Purification of antisera
IgG was purified from the antisera using a combination of ammonia sulfate precipitation and Protein A resin. Briefly, one volume of serum was mixed with an equal volume of 0.15M NaCl. To the mixture, ammonium sulfate (313 g/1) was then added. After 2 hours of stirring, precipitated proteins were obtained by centrifugation (10,000 x g; 10 min), resuspended in a small volume of 0.15M NaCl, and dialyzed against phosphate buffered saline (PBS) . Afterwards, the sample was mixed with one volume of loading buffer (1.5M Glycine, 3M NaCl, pH 8.9) and loaded onto a 5-ml Protein A column. The column was washed with 20 ml of loading buffer and IgG was eluted with 100 mM citric acid, pH 4. The eluate was neutralized with 1 M Tris-HCl, pH 7.4 and dialyzed against PBS. The IgG preparation was then aliquoted and stored at -20°C.
3. Preparation of platelet cytosol Platelets of human, rat or mouse were prepared as previously described (Steinert, et al., Int. J. Cancer 54:92-101 (1993)) . Briefly, blood was mixed with one volume of sodium citrate anticoagulant and centrifuged at 50 x g for 30 minutes. The supernatant was saved. The pellet was mixed with 1/3 volume of platelet wash (Ca2+, Mg2+ and phenol red free MEM) containing 0.8% of 0.1M EDTA and then centrifuged at 50 x g for 20 minutes. The supernatant was saved and the pellet was washed two more times. The supernatants were pooled and centrifuged at 700 x g for 10 minutes. The pellet (washed platelets) was resuspended in phosphate buffered saline to approximately 108/ιπl. To prepare platelet cytosol, platelets were sonicated (4 x 15 sec) in PBS containing 0.5 μM PMSF and 1 μg/ml leupeptin. The sonicate was then centrifuged at 100,000 x g for 60 minutes at 4°C and the supernatant was saved. Protein content was measured by the Bradford dye binding assay using bovine serum albumin as standard.
4. Western blotting Cytosol of human, rat, or mouse platelets (25 μg each) or pure 12-LOX protein (10 ng, 3) was mixed with 2X electrophoresis sample buffer and boiled for 5 minutes. The proteins were separated on a 10% SDS-PAGE gel and then electrophoretically transferred to nitrocellulose membrane.
Non-specific binding sites on the membrane was blocked with BLOTTO (10% non-fat dry milk in PBS containing 0.05% TWEEN-20, Sigma Chemical, St. Louis, MO) . The membrane was then incubated for 1.5 hours with anti 12-LOX IgG diluted 1:1000 in BLOTTO followed by incubation for 1 hour with Horse radish peroxidase conjugated goat anti-rabbit antibody (1:5000 in BLOTTO) . Following extensive wash with PBS containing 0.05% TWEEN-20, the bound secondary antibody was revealed by the ECL Western blot detection system (Amersham Life Science, Little Chalfort, Buckinghamshire, England) . The results were recorded on the Amersham HYPERFILM-ECL. In blocking experiments, 12-LOX antibody (1:1000 in PBS) was preincubated for 60 minutes with human platelet cytosol or bovine serum albumin (50 μg/ml) before incubation with blocked nitrocellulose membrane. Results As shown in Figure 13A, the anti-12-LOX peptide antibody detected a single band of approximately 72 kD in the purified human/HEL 12-LOX sample. It picked up a strong single band in the cytosol from human and mouse platelets, and a much weaker band in cytosol of rat platelets. The molecular weight of the bands in platelet cytosol samples was slightly smaller than that in the purified 12-LOX. When the antibody was preincubated with human platelet cytosol as a source of native human 12-LOX, little immunoreactivity between the antibody and the platelet 12-LOX from human and mouse was observed (Figure 13B) . However, incubation of the 12-LOX antibody with a non-specific protein, BSA, did not render it loss of immunoreactivity to the 12-LOX in the platelet cytosol (Figure 13C) .
Example 5 This example shows an internal control for quantification of 12-LOX mRNA by RT-PCR. As can be seen, RT-PCT is a sensitive and rapid method to detect gene expression. However, there are several inherent weaknesses when use of RT-PCR as a quantification method. RT-PCR results can be affected by conditions for synthesis of DNA molecules from a RNA preparation, effectiveness of DNA molecule amplification during PCR cycles and the richness of target molecules in experimental samples. Therefore, including an internal control during RT-PCR eliminates the variations between experiments as well as subtle differences among individual reaction tubes.
An internal control for the RT-PCR of 12-LOX gene was developed as shown in Figures 14 and 15. A fragment of DNA was cloned into plasmid. The DNA fragment comprises 12-LOX specific sequences at the left and right ends (represented by arrows) . The 12-LOX sequence fragment is flanked by two promoters (signal necessary for synthesis of RNA from DNA) . The construct is introduced into E. coli bacterium and thus replicated to large quantities. The construct is used by synthesis of 12-LOX sequence RNA. A known amount of the 12-LOX sequence RNA is included in each RT-PCR reaction as internal control. Signal derived from the 12-LOX control RNA is easily differentiated from signal derived from 12-LOX gene RNA. 12-LOX gene RNA signal is normalized to 12-LOX control RNA signal. Therefore, quantification of 12-LOX is achieved by referencing the same internal control. In Figure 14, the construct is a plasmid with 12-LOX sequence fragment. The plasmid is pBlueScript SK (Stratagene, LaJolla, CA) . The promoters are SP6 and T7. The fragment sequence is shown in
Figure 15 (SEQ ID NO: 14 and SEQ ID NO: 15) . EXAMPLE 6 Figure 16 shows the presence of enhanced levels of platelet 12-LOX in prostate cancer tissue. One μg of total RNA from matching normal prostate tissue samples and prostate cancer tissue samples from the same patient were used to synthesize cDNA and amplified by PCR as in Example 2. Equal volume of PCR products (15 μl) was separated on a 1.0% agarose gel and transferred, crosslinked, hybridized with a radioactive p32 probe prepared from platelet-type 12 '-LOX cDNA fragment. The membrane was washed under high stringency conditions and exposed to x-ray film at -80°C. Result from 5 pairs of sample is shown. N: normal tissue, T: tumor tissue. The results clearly show the enhanced amounts of 12-LOX in the prostate cancer tissue. Table 3 shows the results in tabular form.
Table 3 Frequency of elevated 12L0X mRNA Expression in Human Prostate Cancer
TNM staging Gleason score grading Race
T2 T3 NO Nl <7_ >8 B
T2 T3 Total
Case 24 39 21 30 51 10 47 16 25 40 Positive 1 19 1 14 15 5 12 7 8 11 Frequency 4% 49; 5% 47% 29% 50% 205 44% 325 28%
T2: confined to gland (non-invasive); T3: extension beyond gland (invasive); NO: no lymph ∞ node metastases; Nl: lymph node metastases present; B: African American; C: Caucasian. ' Positive: more than 50% increase of 12-LOX expression in cancer tissue compared to matching normal tissue.
TNM staging is "Tumor Lymph node metastasis".
Figure imgf000030_0001
Example 7 Detection of elevated 12-lipoxygenase expression -in human prostatic carcinoma with Cytokeratin correction. 12-lipoxygenase (12-LOX) mRNA levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) from matching tumor and normal tissues from same patients. Same initial total RNA loading for cDNA synthesis was controlled by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Tso et al. Nucleic Acids Research, .13, 2485-2502 (1985)) which is ubiquitously expressed in all cell types including both epithelial and stromal cells. Since human prostate cancer cells are of epithelial origin, the best way to correct the difference of cancerous tissue content in tumor and normal tissues is to measure the epithelial cell content in both tumor and normal tissues. Cytokeratin 18 (Leube et al., Differentiation 33:69-85 (1986); Oshi a et al., Differentiation 33:61-68 (1986)) served as an epithelial maker for this purpose for the following reasons: i) . • Cytokeratins including cytokeratin 18 have been shown to be expressed exclusively in cells of epithelial origin (Hallowes et al., Anticancer Research 11: 1799-1806 (1991); Vachon et al., The Anatomical Record 236:567-576 (1993); ii) . Immunochemistry studies using antibodies against cytokeratin 18 showed that normal epithelial cells and cancerous epithelial (carcinoma) cells expressed same levels of cytokeratin 18. Therefore, mRNA expression of 12-LOX by normalization of the amount of the RT-PCR amplified fragments to the GAPDH fragment was calculated and then the value corrected to the cytokeratin 18 expression level. The exclusion criterion for elevated expression of 12-LOX mRNA used in Table 4 is a value larger than 25% increase in 12-LOX from cancer tissue compared to matching normal tissue from same patient. In Table 3 of Example 5 a larger than 50% elevation of 12-LOX was used in tumor tissue verses normal tissue as a cutoff for consideration of positive elevation. With the refinement of the assay (i.e., cytokeratin correction), this value was lowered to >25% elevation. MATERIALS AND METHODS
Tissue specimens. Matching prostatic carcinoma and normal tissues were obtained from 50 patients undergone radical prostatectomy. Tissue samples were immediately frozen at -80°C. A 3-μm section was cut from each tissue and stained with hematoxylin/eosin. Samples containing no carcinoma cells were considered normal and samples containing more than 70% carcinoma cells were considered tumor. Total RNA Isolation. Total RNA from tissues was isolated by guanidinium-isothiocyanate/CsCl gradient centrifugation method with modifications. Briefly, tissue sample up to 0.5g was homogenized in Tempest IQ Homogenizer) in 3.5 ml of guanidinium isothiocyanate (GT) solution containing 4M GT, IM NaAc (pH 5.0), and 0.5M DTT. Then the lysate was transferred into a Ti 55 tube containing 1.3 ml of cesium chloride (CsCl) solution (5.7M CsCl and 0.1M EDTA (pH 6.5)). Centrifuged in Ti 55 rotor (Beckman XL-90 Ultracentrifuge) at 42,000 rpm, 20°C for at least 12 hours. The pellet was suspended in 300 μl of cold 10 mM Tris (pH 8.0). The sample was then phenol- extracted, ethanol-precipitated, and resuspended in distilled water. The optical density was then measured at 260 nm (wavelength) in Pharmacia Ultrospec III Spectrophotometer.
Oligonucleatides synthesis: Oligonucleatides designed for PCR were synthesized in the Gene Assembler Plus (Pharmarcia LKB) . The sequences of the primers used were: 5'-CCT TCC CGT GCT ACC GCT GG-3' (12LOX, sense), 5'-TGG GGT TGG CAC CAT TGA GG-31 (12-LOX antisense), 5'-CAG GAG ACA ATG CTT TGG AC-3' (12-LOX, nested sense) , 5'-GAA CAA CTC ATC ATC ATC ATC CTG CC-3 • (12-LOX nested antisense), 5'-ACT GGC GTC TTC ACC ACC AT-31 (GAPDH, sense), and 5'-TGA CCT TGC CCA CAG CCT TG-3• (GAPDH, antisense), 5•-GAA GAA GAA CCA CGA AGA GGA AG-3' (cytokeratin 18, sense, SEQ ID NO: 16), and 5'-ACT GTG GTG CTC TCC TCA ATC TG-3' (cytokeratin 18, antisense, SEQ ID NO: 17) .
Reverse transcription: One μg of total RNA was reverse transcribed in 20 μl solution containing 50 mM Tris pH 8.3, 75 mM KCl, 3 mM MgCl2, 5 mM DTT, 0.5 mM dNTP, 75 ng/ml of BSA, 30 units of RNasin (Promega) , 2 μM of anti-sense primer, 200 units of MMLV reverse transcriptase (Gibco BRL) at room temperature for 10 min and 37°C for 1 h. Polymerase chain reaction: One μl of reverse transcription mixture was amplified by PCR in 100 μl buffer containing 10 mM Tris pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.1 mg/ml BSA, 1 μM of sense and anti-sense primers and 2.5 unit AmpliTaq DNA polymerase (Perkin Elmer Cetus) . The PCR was run in the GeneAmp PCR system 9600 (Perkin Elmer Cetus) at 94°C, 30 sec; 49- 58°C, 30 sec and 72°C, l min for 25-30 cycles. For nested PCR, 1 μl of first PCR product was reamplified under the same conditions but with nested primers. Same amount of total RNA from different tissues was amplified by RT-PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Tso et al., Nucleic Acids Research, Volume 13, Number 7, 2485-2502 (1985)) and cytokeratin 18 (Leube et al. , Differentiation 33:69-85 (1986); Oshima et al. , Differentiation 33:61-68 (1986) expression using GAPDH and cytokeratin 18 specific primers in separate tubes. GAPDH reaction is used to confirm that equal amounts of total RNA were reverse transcribed (Maier et al., Science, Vol. 249, 1570-1574 (1990); Maier et al. , The Journal of Biological Chemistry, Vol. 265, No. 19, pp. 10805-10808 (1990)), whereas cytokeratin 18 reaction serves as a correction factor for epithelial cell content. The conditions of RT-PCR for GAPDH and cytokeratin 18 were the same as that of 12-LOX. One tenth (10 μl) of PCR products were electrophoresed on 2% agarose gels, which were then visualized by ethidium bromide staining and photographed.
Southern Blotting: Fifteen μl of PCR product was separated on a 1.0% agarose gel and transferred to GeneScreen plus membrane (Du Pont-New England Nuclear) , UV-crosslinked. Radioactive probe was prepared with [32P]dCTP using Prime-it random primer labelling kit (Stratagene) and platelet-type 12-LOX cDNA fragment (a gift from C. D. Funk) . The membrane was prehybridized in the solution containing 50% formamide, , 5 X Denhardt's, 10% dextran sulfate, 5 X SSPE, 0.1% SDS, and lOOμg/ml salmon sperm DNA. Hybridization was performed with the addition of labelled 12-LOX probe (42°C overnight). The membrane was washed under high stringency conditions (0.1 X SSC and 0.1% SDS for 15 minutes at room temperature and three time at 65°C) and exposed to Kodak XOMAT x-ray film at -80°C using an enhancing screen.
DNA sequencing : The Sanger dideoxynucleotide-mediated termination method was used with the Sequenase Version 2.0 DNA sequencing kit (USB, Cleveland, OH) . Plasmid sequencing was done according to the manufacturer's instructions. For direct sequencing of PCR products, the following modifications were done. Agarose gel purified double strand cDNA fragment and primer were denatured at 100°C for 5 minutes and annealed on dry ice for 2 minutes. The DNA chain was labeled using [35S]-dATP and extended on ice for 10 minutes with Sequenase, and terminated at 45°C for 10 minutes. The samples were loaded onto 6% DNA sequencing gel (Base Ace with temperature controller, Stratagene) . The same sample was loaded twice at a 1 hour interval, migrated for a total of 2 hours at 45°C and at an average of 100 watts. The gel was fixed, dried and exposed to x-ray film. DNA sequencing was performed on both strands to confirm the accuracy. The data was recorded by a sequence gel reader (IBI, New Haven, CT) which was connected to MacVECTOR (IBI, New Haven, CT) on a Macintosh Ilci computer.
Gel Densitometry Scanning. The intensity of individual bands was measured by densitometry scanning using an LKB 2400 GelScan XL laser densitometer (Pharmacia, Piscataway, NJ) .
Data Analysis. mRNA expression of 12-LOX was calculated by normalization of the amount of the RT-PCR amplified fragments to the 367 bp GAPDH fragment, then corrected by the cytokeratin 18 expression level.
Table 4
Frequency of elevated 12L0X mRNA Expression in Human Prostate Cancer
TNM staging Gleason score grading Race
T2 T3 NO Nl <7 >8 B
T2 T3
Case 24 39 21 30 10 47 16 25 40 Positive 1 25 1 19 6 13 11 14 13 Frequency 4% 64% 5% 63 % 60% 28% 69 % 56^ 33 ^
I bo
T2: confined to gland (non-invasive); T3: extension beyond gland (invasive); NO: no lymph ^ node metastases; Nl: lymph node metastases present; B: African American; C: Caucasian. Positive: more than 25% increase of 12-LOX expression in cancer tissue compared to matching normal tissue.
It is intended that the foregoing description be only illustrative of the present invention and that the present invention be limited only by the hereinafter appended claims.
APPENDIX I
(1) GENERAL INFORMATION:
(i) Applicant: Kenneth V. Honn, Yong Q. Chen and John D. Crissman (ii) Title of Invention: Methods for Detecting
12-Lipoxygenase and Its Encoding DNA (iii) Number of Sequences: 17 (iv) Correspondence Address:
(A) Addressee: Ian C. McLeod
(B) Street: 2190 Commons Parkway
(C) City: Okemos
(D) State: Michigan
(E) Country: USA
(F) Zip: 48864
(v) Computer Readable Form:
(A) Medium Type: Diskette 5.25 inch, 360 kb storage
(B) Computer: Acer
(C) Operating System: MS-DOS (version 3.3)
(D) Software: WordPerfect 5.1 (vi) Current Application Data:
(A) Application Number: 08/212,135
(B) Filing Date: March 14, 1994
(C) Classification: (vii) Prior Application Data:
(A) Application Number:
(B) Filing Date:
(viii) Attorney/Agent Information:
(A) Name: Ian C. McLeod
(B) Registration No.: 20,931
(C) Reference/Docket Number: Biomide
4.1-1 (ix) Telecommunication Information:
(A) Telephone: (517) 347-4100 (B) Telefax: (517) 347-4103 (2) Information for SEQ ID NO: 1
(i) Sequence Characteristics:
(A) Length: 20 Bases
(B) Type: Nucleic Acid
(C) Strandedness: Single
(D) Topology: Linear (ii) Molecule Type:
(A) Description: DNA primer for segment of 12-lipoxygenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) Organism: human (vii) IMMEDIATE SOURCE:
(A) Library: Genomic (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1
CCTTCCCGTG CTACCGCTGG 20
(3) Information for SEQ ID NO: 2
(i) Sequence Characteristics:
(A) Length: 20 Bases
(B) Type: Nucleic Acid
(C) Strandedness: Single
(D) Topology: Linear (ii) Molecule Type:
(A) Description: DNA primer for a segment of 12-lipoxygenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE: (A) Library: Genomic (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CAGGAGACAA TGCTTTGGAC 20
(4) Information for SEQ ID NO: 3
(i) Sequence Characteristics:
(A) Length: 20 Bases
(B) Type: Nucleic Acid
(C) Strandedness: Single
(D) Topology: Linear (ii) Molecule Type:
(A) Description: DNA primer for a segment of 12-lipoxygenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (vi) ORIGINAL SOURCE:
(A) Organism: (vii) IMMEDIATE SOURCE:
(A) Library: Genomic (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TGGGGTTGGC ACCATTGAGG 20
(5) Information for SEQ ID NO: 4
(i) Sequence Characteristics:
(A) Length: 20 Bases
(B) Type: Nucleic Acid
(C) Strandedness: Single
(D) Topology: Linear ( ii ) Molecule Type :.
(A) Description: DNA primer for a segment of 12-lipoxygenase (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE:
(A) Library: Genomic (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GAACAACTCA TCATCCTGCC 20
(6) Information for SEQ ID NO: 5
(i) Sequence Characteristics:
(A) Length: 20 Bases
(B) Type: Nucleic Acid
(C) Strandedness: Double
(D) Topology: Linear (ii) Molecule Type:
(A) Description: DNA encoding a segment of 12-lipoxygenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE:
(A) Library: Genomic (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
C TCC GGG TCG TAC AAC CGC G 20 Ser Gly Ser Thr Asn Arg
5
(7) Information for SEQ ID NO: 6
(i) Sequence Characteristics:
(A) Length: 24 Base Pairs
(B) Type: Nucleic Acid
(C) Strandedness: Double
(D) Topology: Linear (ii) Molecule Type:
(A) Description: DNA encoding a segment of 12-lipoxygenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE:
(A) Library: Genomic
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CCA GGA GAC AAT GCT TTG GAC ATG 24 Pro Gly Asp Asn Ala Leu Asp Met
5
(8) Information for SEQ ID NO: 7
(i) Sequence Characteristics:
(A) Length: 24 Base Pairs
(B) Type: Nucleic Acid
(C) Strandedness: Double
(D) Topology: Linear (ii) Molecule Type:
(A) Description: Genomic DNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE:
(A) Library: Genomic
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
ACC ATC GCT .GCA GAC CGT AAG GAT 24 Thr lie Ala Ala Asp Arg Lys Asp
5
(9) Information for SEQ ID NO: 8
(i) Sequence Characteristics:
(A) Length: 57 Base Pairs
(B) Type: Nucleic Acid
(C) Strandedness: Double
(D) Topology: Linear (ii) Molecule Type:
(A) Description: DNA encoding a segment of 12-lipoxygenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE:
(A) Library: Genomic (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
TGG ACA CTG AAG GCA GGG GCT CTG GAG ATG GCC 33 Trp Thr Leu Lys Ala Gly Ala Leu Glu Met Ala
5 10
CTC AAA CGT GTT TAC ACC CTC CTG 57
Leu Lys Arg Val Tyr Thr Leu 15
(10) Information for SEQ ID NO: 9
(i) Sequence Characteristics:
(A) Length: 24 Base Pairs
(B) Type: Nucleic Acid
(C) Strandedness: Double
(D) Topology: Linear (ii) Molecule Type:
(A) Description: DNA encoding a segment of 12-lipoxygenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE:
(A) Library: Genomic
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CTC CCA GGT GCT CTC TAT GCC CAT 24 Leu Pro Gly Ala Leu Thr Ala His
5
(11) Information for SEQ ID NO: 10
(i) Sequence Characteristics:
(A) Length: 19 Bases
(B) Type: Nucleic Acid
(C) Strandedness: Single
(D) Topology: Linear (ii) Molecule Type:
(A) Description: DNA primer for a segment encoding glycero- aldehyde-3-phosphate dehydrogenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE:
(A) Library: Genomic (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ACT GGC GTC TTC ACC ACC AT 19
(12) Information for SEQ ID NO: 11
'i) Sequence Characteristics:
(A) Length: 20 Bases
(B) Type: Nucleic Acid
(C) Strandedness: Single
(D) Topology: Linear (ii) Molecule Type:
(A) Description: DNA primer for a segment encoding glyceraldehyde-3- phosphate dihydrogenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE:
(A) Library: Genomic (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GA CCT TGC CCA CAG CCT TG 20
(13) Information for SEQ ID NO: 12
(i) Sequence Characteristics:
(A) Length: 21 Bases
(B) Type: Nucleic Acid
(C) Strandedness: Single
(D) Topology: Linear (ii) Molecule Type:
(A) Description: DNA primer for 12- lipoxygenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE:
(A) Library: Genomic (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CGC GGT TGT ACG ACC CGG AGU 21
(14) Information for SEQ ID NO: 13
(i) Sequence Characteristics:
(A) Length: 21 Bases
(B) Type: Nucleic Acid
(C) Strandedness: Single
(D) Topology: Linear (ii) Molecule Type: (A) Description: DNA primer for 12-lipoxygenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE: (A) Library: N/A ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CTC CGG GTC GTA CAA CCG CGU 21
(15) Information for SEQ ID NO: 14
(i) Sequence Characteristics:
(A) Length: 160 Bases
(B) Type: Nucleic Acid
(C) Strandedness: Single
(D) Topology: Linear (ii) Molecule Type:
(A) Description: Segment of 12-lipoxygenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE: (A) Library: N/A (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: AGCTTCCTTC CCGTGCTACC GCTGGGTGGG GCGCCCTAGG CACCACAGGA 50 GACAATGCTT TGGACCCATG GTTTTGTTGG CCATGTTATC CATGGTCTGG 100 ATCGGCGGCT CCATCGGCAG GATGATGAGT TGTtCCCTCA ATGGTGCCAA 150 CCCCACTGCA 160
(16) Information for' SEQ ID NO: 15
(i) Sequence Characteristics:
(A) Length: 152 Bases
(B) Type: Nucleic Acid
(C) Strandedness: Single
(D) Topology: Linear (ii) Molecule Type:
(A) Description: Segment of 12-lipoxygenase. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE:
(A) Library: N/A (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
AGGAAG GGCACGATGG CGACCCACCC CGCGGGATCC GTGGTGTCCT 46
CTGTTACGAA ACCTGGGTAC CAAAACAACC GGTACAATAG GTACCAGACC 96
TAGCCGCCGA GGTAGCCGTC CTACTACTCA ACAAGGGAGT TACCACGGTT 146
GGGGTG 152
(17) Information for SEQ ID NO: 16
(i) Sequence Characteristics:
(A) Length: 23 Bases
(B) Type: Nucleic Acid
(C) Strandedness: Single (D) Topology: Linear (ii) Molecule Type:
(A) Description: Segment of DNA for cytokeratin. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE: (A) Library: N/A (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GAA GAA GAA CCA CGA AGA GGA AG 23
(18) Information for SEQ ID NO: 17
(i) Sequence Characteristics:
(A) Length: 23 Bases
(B) Type: Nucleic Acid (C) ' Strandedness: Single (D) Topology: Linear
(ii) Molecule Type:
(A) Description: segment DNA for cytokeratin. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (vi) ORIGINAL SOURCE:
(A) Organism: Human (vii) IMMEDIATE SOURCE:
(A) Library: N/A (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: ACT GTG GTG CTC TCC TCA ATC TG 23

Claims

WE CLAIM:
-1- A method for detecting an RNA for 12- lipoxygenase from cells which comprises:
(a) extracting the RNA from the cells;
(b) binding the RNA with spaced apart sense and antisense primers defining a region of the DNA of the 12-lipoxygenase which is unique;
(c) reverse transcribing the RNA bound with the sense and the antisense primers with a polymerase to provide a cDNA from the RNA; and (d) detecting the RNA for the 12- lipoxygenase from the cDNA.
-2-
A method for detecting an RNA for 12- lipoxygenase from cells which comprises:
(a) extracting the RNA from the cells;
(b) binding the RNA with a sense primer selected from the group consisting of
5 •-CCTTCCCGTGCTACCGCTGG-3 ' and 5«-CAGGAGACAATGCTTTGGAC-3 ' as set forth in sequence ID NOS. 1 and 2 and an antisense primer selected from the group consisting of: 5-TGGGGTTGGCACCATTGAGG-3 ' and
5-GAACAACTCATCATCCTGCC-3 • as set forth in SEQ ID NOS. 3 and 4;
(c) reverse transcribing the RNA bound with the sense and the antisense primers with a polymerase to provide a cDNA from the RNA; and
(d) detecting the RNA for the 12- lipoxygenase from the cDNA. -3- The method of Claim 2 wherein the cDNA is detected by labeling the cDNA and then an amount of the label is determined.
-4- The method of Claim 2 wherein the cDNA is detected by binding the cDNA with a labeled segment of a peptide or DNA selected from the group consisting of SEQ ID NO: 1 to 9, 12 and 13 and an amount of the label is determined.
-5- The method of Claim 2 wherein the cDNA in step (d) is removed from the RNA, separated by gel electrophoresis and then is compared to a known standard sample of the cDNA.
-6- A DNA which is a segment of 12-lipoxygenase and selected from the group consisting of SEQ ID NOS. 1 to 9, 12 and 13 free of other DNA.
-7- A peptide which is a segment of human 12- lipoxygenase and derived from the group consisting of SEQ ID NOS. 5, 6, 7, 8, and 9, substantially free of other peptides.
-8- A method for detecting 12-lipoxygenase or a 12-lipoxygenase RNA which comprises:
(a) binding the 12-lipoxygenase or RNA with a labeled DNA selected from the group consisting of SEQ ID NOS: 1 to 9, 12 and 13; and
(b) detecting the DNA bound 12-lipoxygenase or RNA. -9- The method of Claim 8 wherein the label is a radiolabel.
-10- The method of Claim 8 wherein the label is an enzyme label.
-11- A DNA primer selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 13 and 14.
-12- A method for detecting 12-lipoxygenase which comprises:
(a) reacting a Western Blot containing cellular 12-lipoxygenase with an antibody to the 12- lipoxygenase derived from SEQ ID NO: 8 to form a complex of the 12-lipoxygenase and the antibody; and
(b) detecting the complex.
-13- The method of Claim 12 wherein the antibody is a polyclonal antibody.
-14- The method of Claim 13 wherein the antibody is derived from a rabbit.
-15- The method of Claim 12 wherein a labeled anti-antibody is reacted with the complex.
-16- The method of Claim 15 wherein the anti- antibody is labeled with a peroxidase and is detected by a reaction with hydrogen peroxide and luminol to produce chemiluminescence.
-17- In a method for detecting an RNA of 12- lipoxygenase by reverse transcribing the RNA and detecting a first cDNA produced by the reverse transcribing, the improvement which comprises: providing as an internal standard with the RNA a segment of the RNA which is reversed transcribed and detected as a second cDNA along with the first cDNA. -18- The method of Claim 17 wherein the second cDNA is as set forth in SEQ ID NO: 14 and SEQ ID NO: 15.
-19- The method of Claim 17 wherein the first and second cDNA is detected in the method for detecting an RNA for 12-lipoxygenase from cells which comprises: (a) extracting the RNA from the cells; (b) binding the RNA with a sense primer selected from the group consisting of 5'-CCTTCCCGTGCTACCGCTGG-3 ' and 5'-CAGGAGACAATGCTTTGGAC-3 ' as set forth in sequence ID NOS. 1 and 2 and an antisense primer selected from the group consisting of: 5-TGGGGTTGGCACCATTGAGG-3 ' and
5-GAACAACTCATCATCCTGCC-3 ' as set forth in SEQ ID NOS. 3 and 4;
(c) reverse transcribing the RNA bound with the sense and the antisense primers with a polymerase to provide a cDNA from the RNA; and
(d) detecting the RNA for the 12- lipoxygenase from the cDNA.
-20- The method of Claim 19 wherein the cDNA is detected by labeling the cDNA and then an amount of the label is determined.
-21-
The method of Claim 19 wherein the first and second cDNA is detected by binding the cDNA with a labeled segment of a peptide or DNA selected from the group consisting of SEQ ID NO: l to 9, 12 and 13 and an amount of the label is determined. -22- The method of Claim 19 wherein the first and second cDNA in step (d) is removed from the RNA, separated by gel electrophoresis and then is compared to a known standard sample of the first and second cDNA.
-23- A DNA substantially free of other DNA comprising a sequence sufficiently duplicative of that set forth in the group consisting of SEQ ID NOS: 5, 6, 7, 8 and 9 such that the DNA selectively hybridizes under stringent conditions to DNA encoding 12-LOX.
-24- The DNA of Claim 23 wherein there is at least about 80 percent homology.
-25-
A peptide free of other peptides and allelic or species variants thereof which is a segment of 12- lipoxygenase and derived from a DNA substantially free of other DNA comprising a sequence sufficiently duplicative of that set forth in the group consisting of SEQ ID NOS: 5, 6, 7, 8 and 9 such that the DNA selectively hybridizes under stringent conditions to DNA encoding 12-LOX.
-26-
The method of Claim 1 wherein the RNA is extracted from prostate cells including stromal and epithelial cells and wherein the RNA for 12- lipoxygenase which is only expressed in epithelial cells as verified by the in situ data is determined. -27- The method of Claim 27 wherein the epithelial cells are determined by a cytokeratin present in the cells using a polymeric chain reaction.
-28- The method of Claim 27 wherein the primers for cytokeratin are as set forth in SEQ ID NOS: 16 and 17.
-29-
A method for detecting 12-lipoxygenase or a 12-lipoxygenase RNA which comprises: binding the 12-lipoxygenase or RNA with a labeled DNA substantially free of other DNA comprising a sequence sufficiently duplicative of that set forth in the group consisting of SEQ ID NOS: 5, 6, 7, 8 and 9 such that the DNA selectively hybridizes under stringent conditions to DNA encoding 12-LOX.
-30- The DNA of Claim 29 wherein there is at least 80 percent homology.
PCT/US1995/001623 1994-03-14 1995-02-06 Methods for detecting 12-lipoxygenase and its encoding dna WO1995025178A1 (en)

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WO1999013111A1 (en) * 1997-09-11 1999-03-18 Abbott Laboratories Reagents and methods useful for detecting diseases of the prostate
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JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 268, No. 1, issued 05 January 1993, SHORNICK et al., "A Cryptic, Microsomal-Type Arachidonate 12-Lipoxygenase is Tonically Inactivated by Oxidation-Reduction Conditions in Cultured Epithelial Cells", pages 371-376. *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999013111A1 (en) * 1997-09-11 1999-03-18 Abbott Laboratories Reagents and methods useful for detecting diseases of the prostate
US6103496A (en) * 1998-05-29 2000-08-15 Vanderbilt University Isolated and purified 12R-lipoxygenase protein and nucleic acids
US6569644B2 (en) 1998-05-29 2003-05-27 Vanderbilt University Isolated and purified 12R-lipoxygenase protein and nucleic acids

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