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WO1996041003A1 - Detection du facteur de risque du cancer colorectal et traitement - Google Patents

Detection du facteur de risque du cancer colorectal et traitement Download PDF

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Publication number
WO1996041003A1
WO1996041003A1 PCT/US1996/009009 US9609009W WO9641003A1 WO 1996041003 A1 WO1996041003 A1 WO 1996041003A1 US 9609009 W US9609009 W US 9609009W WO 9641003 A1 WO9641003 A1 WO 9641003A1
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Prior art keywords
pla2s
individual
protein
colon cancer
mutation
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PCT/US1996/009009
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English (en)
Inventor
Arthur M. Buchberg
Linda D. Siracusa
Kenneth P. Chepenik
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Thomas Jefferson University
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Publication of WO1996041003A1 publication Critical patent/WO1996041003A1/fr

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    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01004—Phospholipase A2 (3.1.1.4)
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)

Definitions

  • the invention relates to genetic alterations associated with colon cancer.
  • the invention relates to the discovery of genetic mutations that are associated with increased risks of familial and spontaneous colon cancer.
  • the invention relates to prophylactic compositions and method of preventing colon cancer.
  • Colon cancer is a major cause of death in the United States. Despite progress in the treatment of many forms of cancer, colon cancer continues to be responsible for deaths in the United States.
  • APC Adomatous Polyposis Coli
  • Germ-line mutations of APC are found in inherited familial cancers such as Gardner's syndrome, attenuated adenomatous polyposis coli, heredity flat adenoma syndrome and familial adenomatous polyposis (FAP) .
  • FAP familial adenomatous polyposis
  • APC adenomatous polyps in the second to third decades of life, which if left untreated progress to malignant carcinomas.
  • Genetic linkage analysis localized the APC gene to human chromosome 5q21-q22, a region frequently associated with allelic loss of the wildtype 5q allele. Mutations in APC are also implicated in sporadic colorectal cancers and in extracolonic tumors, such as gastric and small intestinal polyps, osteomas, sarcomas and desmoidal tumors.
  • the present invention relates to methods of identifying an individual at an elevated risk of colon cancer.
  • the methods comprise the steps of isolating genetic material from a tissue or body fluid sample from said individual and detecting a PLA2s gene mutation.
  • a PLA2s mutation is indicative that the individual is at an elevated risk of colon cancer.
  • the present invention relates to methods of identifying an individual at an elevated risk of colon cancer.
  • the methods comprise the steps of isolating protein from a tissue or body fluid sample from said individual and detecting an absence of PLA2s protein or PLA2s enzyme activity in said isolated protein sample. The absence of PLA2s protein or PLA2s enzyme activity in said isolated protein sample is indicative that the individual is at an elevated risk of colon cancer.
  • the present invention relates to methods of preventing colon cancer or reducing risk of colon cancer in an individual who is identifying as being at an elevated risk of colon cancer.
  • the methods comprise the steps of identifying an individual predisposed to colon cancer by detecting a PLA2s gene mutation or PLA2s protein or enzyme activity deficiency and administering to said individual a prophylactically effective amount of phospholipase protein or a recombinant vector comprising a nucleotide sequence that encodes PLA2s.
  • the present invention relates to a transgenic animal which comprises a transgene including a nucleotide sequence that encodes PLA2s.
  • the parental strain of the transgenic mice is null for PLA2s.
  • the present invention relates to methods of preventing colon cancer or reducing risk of colon cancer in an individual who is identifying as being at an elevated risk of colon cancer due to a PLA2s deficiency.
  • the methods comprise the steps of identifying an individual predisposed to colon cancer by detecting a PLA2s protein or enzyme activity deficiency and administering to said individual a prophylactically effective amount of compound that upregulates endogenous PLA2s expression.
  • the PLA2s gene maps to mouse chromosome 4.
  • Panel A Haplotype analysis of the loci mapped in the interspecific backcross are shown. The loci mapped are listed to the left of each row with the most proximal locus listed first. A total of 167 N 2 progeny were typed for all the markers. Each column represents the chromosome identified in the N2 offspring that was inherited from the F 1 parent. Black squares represent the AEJ/Gn allele. White squares represent the Mus spretus allele. The number of N 2 progeny carrying each type of chromosome is listed below each column. Panel B. Loci were placed on the map based on the results of the interspecific backcross analysis (left side) .
  • Panel A Illustrates the strains that exhibit high levels of PLA2s expression, with C57BL/6J(B6) mice included as a control.
  • Panel B Illustrates the strains that exhibit low levels of PLA2s expression, with AKR/J (AKR) mice included as a control. Strains that expressed high levels of PLA2s, consistently had higher expression levels in the small intestine in comparison to the large intestine. The 2.6 kb transcript is of unknown origin and was consistently detected in strains expressing PLA2s.
  • FIG. 3 RFLP analysis of the PLA2s gene reveals a BamHI polymorphism that is concordant with expression levels.
  • Genomic DNA (5.0 g per lane) from 11 inbred strains was digested with BamHI. The DNA was electrophoresed in a 0.8% agarose gel and transferred to a Hybond N+ filter. Hybridization was carried out under high stringency with a 32p - dCTP labeled mouse PLA2s cDNA carrying the entire coding sequence. The sizes of the identified restriction fragments are shown.
  • FIG. 4 The sequence of the Mouse PLA2s cDNA.
  • the nucleotide sequence and predicted amino acid sequence of mouse PLA2s is shown.
  • the pairs of black boxes above the nucleotide sequence denote intron/exon junctions as determined by sequencing a genomic close.
  • the mouse PLA2s gene is encoded by five exons.
  • the single underlined region indicates the signal sequence that is removed from the mature product.
  • the boxed sequence denotes the BamHI site that is mutated in Moml s strains; the thymidine insertion is shown within the box.
  • the predicted amino acid sequence of wildtype PLA2s is shown immediately below the nucleotide sequence.
  • the two predicted protein products of the Moml susceptible strains is shown from the site of the thymidine insertion.
  • the dashed amino acid sequence, (labelled B6alt) is the predicted peptide derived from the splicing of exon 3 into exon 5, thus the gap encompassing exon 4 (see Figure 5) .
  • the dotted amino acid sequence (labelled B6) is the truncated product generated from the correctly spliced mRNA.
  • RT-PCR analysis of intestinal RNA reveals the presence of PLA2s in all strains and an alternatively spliced product in Moml s strains.
  • RT-PCR products derived from total RNA isolated from the small intestine of 9 inbred strains listed at the top. PCR products were electrophoresed on 2.0% agarose gels and visualized with ethidium bromide.
  • the primers used for PCR were 5' -GAAACCATACCACCATCCAA-3' (SEQ ID NO:l) and 5'- CCAGGACTCTCTTAGGTACG-3' (SEQ ID NO:2) which amplified nucleotides 11 to 747 of the murine PLA2s cDNA ( Figure 4) .
  • the size marker is the 1 kb ladder (Gibco-BRL, Gaithersburg, MD) , with some sizes shown for orientation located to the right of the gel.
  • the negative control is the same components as all other lanes except no reverse transcriptase was added.
  • the results reveal that all strains contain PLA2s mRNA.
  • the results also demonstrate that all strains carrying the Moml s allele of PLA2s exhibit a novel RT-PCR product.
  • Panel A cDNA sequence surrounding the polymorphic BamHI derived from B6, MA/MyJ and AKR intestinal RNA.
  • the thymidine insertion in the B6 strain is indicated by the arrowhead.
  • RT-PCR products were purified from a 2.0% low melt agarose gel and sequenced.
  • the primer used for sequencing the polymorphic region was 5' -GCGCAGTTTGGGGAAAT-3' (SEQ ID NO:3) corresponding to bp 113 to 130 of the PLA2s cDNA.
  • Panel B Sequence of the novel RT-PCR product detected in B6 intestinal RNA.
  • the nucleotide sequence surrounding the novel junction between exon 3 and exon 5 identified in B6 mice is shown. The different exons are denoted by brackets.
  • Panel C The presence of the BamHI polymorphism is confirmed in genomic DNA.
  • PCR products were generated from genomic DNA from B6 and AKR mice. 100 ng of genomic DNA was amplified with primers 5'-GAGAGCTGACAGCATGAAGG-3' (SEQ ID NO:4) and 5'-CCGTTTCTGACAGGAGTTCTGGTT-3' (SEQ ID NO:5) corresponding to bp 31 and 368 of the PLA2s cDNA.
  • Figure 7 shows the human PLA2s gene sequence.
  • Figure 8 shows a list of restriction enzymes that cut within and outside of the coding sequence.
  • PLA2s type II non-pancreatic phospholipase A2
  • Min murine form of the APC gene
  • PLA2s is a low molecular weight (14 kDa) group II phospholipase belonging to a diverse family of enzymes that hydrolyze the sn-2 fatty acyl ester bond of phosphoglycerides to produce free fatty acids and lysophospholipids.
  • PLA2s is one of the enzymes involved in the production of arachidonic acid, which is the rate limiting substrate for the production of leukotrienes and prostaglandins. Additionally, PLA2s has been implicated in the pathophysiology of acute pancreatitis, rheumatoid arthritis and asthma. High levels of PLA2s expression have been detected in rat and murine small intestine.
  • the PLA2s enzyme has been localized to Paneth cells at the base of the crypts of Lieberkuhn. Expression of this enzyme coincides with the appearance of mature Paneth cells, suggesting the presence of PLA2s during the establishment and maintenance of the villus epithelium.
  • the catalytic domain of PLA2s is at His48.
  • the calcium binding domain is N-terminal to that the His 48 residue.
  • cysteine residues throughout the protein which are involved in the formation of disulfide bridges necessary for the active conformation of the protein. Mutations in PLA2s coding sequences which will effect calcium binding and/or His48 and/or disulfide bridge formation will result in the decrease or elimination of enzyme activity. Accordingly, substitution mutations in the PLA2s coding sequences which result in amino acid residue changes that effect calcium binding and/or His48 and/or disulfide bridge formation will result in the decrease or elimination of enzyme activity.
  • APC mutation Individuals who have been identified as being at risk of colon cancer due to an APC mutation are at an additionally higher risk if identified as also having mutations in one or both PLA2s genes.
  • the combination of APC mutation with a homozygous mutation in PLA2s renders the individual at being at a higher elevated risk.
  • Individuals who have been identified as having normal APC are at an higher risk of colon cancer if identified as having mutations in one or both PLA2s genes.
  • identification of mutations in the PLA2s gene indicates elevated risk of colon cancer.
  • methods, kits and reagents are provided which can be used to screen individuals to determine if they belong in an elevated risk group for colon cancer.
  • a homozygous mutation in the PLA2s gene indicates the individual has an extremely elevated risk of familial colon cancer.
  • a heterozygous mutation in the PLA2s gene indicates the individual has an elevated risk of familial colon cancer.
  • a homozygous mutation in the PLA2s gene indicates the individual has an elevated risk of spontaneous colon cancer.
  • a heterozygous mutation in the PLA2s gene indicates the individual has a somewhat elevated risk of spontaneous colon cancer.
  • Identifying individuals with PLA2s mutations and thereby determining whether individuals are at high risk is useful in the health management of such individuals.
  • Individuals identified as being at genetically higher risk of colon cancer can be monitored extensively and more often than is prescribed for individuals not genetically predisposed to colon cancer.
  • individuals identified as being at higher risk of colon cancer can take preventative steps and treatments to reduce and minimize the risk of colon cancer.
  • Mutations which can reduce or eliminate the production of functioning PLA2s include: mutations in the regulatory regions that facilitate gene expression include alterations of sequence or genetic rearrangement and point mutations within the coding region such as a deletion or substitution which causes a frameshift in the reading frame and the production of a wholly or partially inactive enzyme. Such mutations may be identified by a variety of well known techniques.
  • the lack of production of mRNA may be detected, for example, using Northern blot analysis or RT-PCR.
  • the lack of production of normal, functional PLA2s protein may be detected, for example, using Western blot analysis, immunoassay or enzyme activity assays.
  • Genomic DNA may be analyzed to detect mutations by Southern blot analysis including restriction fragment length polymorphism analysis, oligonucleotide hybridization using allele specific probes, direct nucleotide sequencing of PCR amplified regulatory and coding sequences, single strand conformation polymorphism (SSCP) using PCR amplified regulatory and coding sequences and fragment size analysis of PCR amplified microsatellite DNA.
  • Southern blot analysis including restriction fragment length polymorphism analysis, oligonucleotide hybridization using allele specific probes, direct nucleotide sequencing of PCR amplified regulatory and coding sequences, single strand conformation polymorphism (SSCP) using PCR amplified regulatory and coding sequences and fragment size analysis of PCR amplified microsatellite DNA.
  • SSCP single strand conformation polymorphism
  • Assays can be designed using these techniques to detect the presence or absence of normal PLA2s genes, mRNA and protein as well as the presence or absence of mutated forms of PLA2s genes, mRNA and protein.
  • assays can be designed for quantitative analysis of PLA2s mRNA, protein and enzyme activity.
  • the techniques for performing these analyses are well known to those having ordinary skill in the art and are generally described in Sambrook et al, eds. "Molecular Cloning: A Laboratory Manual” 2nd Edition, Cold Spring Harbor Laboratory Press 1989, which is incorporated herein by reference.
  • the PLA2s gene is published in two segments as Genbank as accession numbers: M22429 J0704 and M22431 J0704, which are incorporated herein by reference. These segments are shown in Figure 7.
  • RNA may be extracted from tissue samples such as biopsy samples and analyzed by Northern blot analysis. Using probes which hybridize to PLA2s mRNA, Northern blot analysis provides the means to determine the presence or absence of mRNA that hybridizes to PLA2s specific probes. The absence of such mRNA is indicative of an absence of PLA2s expression. Known quantities of PLA2s mRNA are used as controls.
  • the present invention relates to kits for performing Northern blot analysis which comprise an container having a PLA2s specific probe and instructions for performing the assay.
  • positive and/or negative controls may be provided and/or representative photos or diagrams of positive and/or negative results.
  • RNA is extracted from tissue samples such as biopsy samples and used in as substrate together with PLA2s specific PCR primers in PCR reactions. If no nucleic acid molecules are amplified, the lack of PLA2s mRNA is indicated. The absence of such mRNA is indicative of an absence of PLA2s expression.
  • Known quantities of PLA2s mRNA are used as controls.
  • the present invention relates to kits for performing RT-PCR analysis which comprise an container having a PLA2s specific primers and instructions for performing the assay.
  • positive and/or negative controls may be provided and/or representative photos or diagrams of positive and/or negative results.
  • Assays to detect the presence and/or quantity of PLA2s protein including assays to detect the presence and/or quantity of PLA2s enzyme activity are also useful to identify individuals at increased risk of colon cancer.
  • Protein is extracted from tissue samples such as biopsy samples or fecal samples and analyzed to detect the presence and/or quantity of proteins that cross react with PLA2s specific antibodies or to detect the presence and/or quantity of PLA2s enzyme activity.
  • protein is acid purified from tissue samples.
  • acid extraction is performed using the method of Franson RC, et al . 1983, Solubilization and characterization of a neutral-active, calcium-dependent, phospholipase A 2 from rabbit heart and isolated chick embryo myocytes. Lung 160:275-284, which is incorporated herein by reference. Known quantities of functional PLA2s are used as controls.
  • Western blot analysis and immunoassay are useful to detect the presence of protein that binds to PLA2s specific antibodies.
  • Western blot analysis is a technique whereby the presence of protein which hybridizes to PLA2s specific antibodies can be detected and analyzed for size. Immunoassay do not measure protein size.
  • Western blot and immunoassay will not detect the presence of proteins encoded by frameshifted genes if the antibodies recognize epitopes eliminated by the frame shift. Thus, antibodies which bind to epitopes on PLA2s which are eliminated in frameshift PLA2s mutations are useful to identify PLA2s mutations.
  • Western blot and immunoassay are performed using antibodies that specifically bind to epitopes that include the catalytic domain of PLA2s, particularly epitopes that include His48. Further, Western blot and immunoassay may be performed using antibodies that specifically bind to epitopes that include the calcium binding domain. Alternately, Western blot and immunoassay are performed using antibodies that specifically bind to epitopes that present due to the formation of disulfide bridges necessary for the active conformation of the protein.
  • antibody is meant to refer to complete, intact antibodies, and Fab fragments and F(ab) 2 fragments thereof.
  • Complete, intact antibodies include monoclonal antibodies such as murine monoclonal antibodies, chimeric antibodies and humanized antibodies.
  • the production of antibodies and the protein structures of complete, intact antibodies, Fab fragments and F(ab) 2 fragments and the organization of the genetic sequences that encode such molecules are well known and are described, for example, in Harlow, E. and D. Lane (1988) ANTIBODIES : A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. which is incorporated herein by reference. Briefly, for example, the PLA2s protein, or an immunogenic fragment thereof is injected into mice.
  • the spleen of the mouse is removed, the spleen cells are isolated and fused with immortalized mouse cells.
  • the hybrid cells, or hybridomas, are cultured and those cells which secrete antibodies are selected.
  • the antibodies are analyzed and, if found to specifically bind to the PLA2s protein, the hybridoma which produces them is cultured to produce a continuous supply of antibodies.
  • kits for performing Western blot analysis or immunoassay which comprise an container having a PLA2s specific antibody and instructions for performing the assay. Additional immunoassay reagents may optionally be provided as described below. Optionally, positive and/or negative controls may be provided and/or representative photos or diagrams of positive and/or negative results.
  • PLA2s enzyme activity assays are well known such as those methods taught in Marshall L.A. et al . , J. Lipid Mediators Cell Signalling 10:295-313 (1994), which is incorporated herein by reference.
  • PLA2s enzyme activity assays can be used to detect and/or quantify the presence of function PLA2s protein in a sample. Protein is extracted from tissue samples such as biopsy samples or fecal samples and analyzed to determine the presence and/or amount of activity to process PLA2s substrate. Known quantities of functional PLA2s are used as controls.
  • Genome DNA may be employed to detect mutations on the PLA2s gene. Some such analyses can be performed directly on extracted genomic DNA to identify genetic rearrangements of the PLA2s gene. Other analyses include the amplification of the exons and adjacent sequences followed by analysis of the amplified DNA. In each case, mutations in the PLA2s gene can be identified.
  • Southern blot analysis may be employed on extracted genomic DNA to identify the presence of nucleotide sequences that hybridize to PLA2s specific probes and for performing restriction fragment length polymorphism analysis to determine if a mutation involving the PLA2s gene results in the elimination of restriction enzyme sites within the coding sequence, or creation of a restriction enzyme site normally not present, or a genetic rearrangement in which the a chromosomal mutation results in the relocation of the PLA2s gene within the genome.
  • Figure 8 shows a list of restriction enzymes that cut within and outside of the coding sequence. These restriction enzymes can be used in RFLP analysis to identify polymorphisms that indicate mutations in the PLA2s gene.
  • restriction enzymes listed as cutting within the coding sequence are particularly useful to identify polymorphisms that indicate mutations with the PLA2s coding sequences that can be used in RFLP analysis. Further, restriction enzymes not listed can be used to identify the creation of new restriction sites due to mutations.
  • polymorphisms are detected by RFLP analysis, the location of the polymorphism can be determined by comparison with the known sequence, using controls as well as using probes that detect smaller regions of the gene. If a polymorphism is detected that is not located within the open reading frame, these polymorphisms provide epidemiological data which is useful to correlate the different alleles of PLA2s as defined by the polymorphisms to the phenotype of individuals carrying them, i.e. the incidence of colon cancer for carriers of the different alleles. Any difference detected can result in subtle expression differences that can contribute to disease risk or protection. Polymorphisms/alleles of PLA2s can be detected by a variety of procedures described herein including Southern blots, SSCP, SSLP etc.
  • the present invention relates to kits for performing
  • Southern blot analysis including RFLP which comprise an container having a PLA2s specific probe and instructions for performing the assay.
  • positive and/or negative controls may be provided and/or representative photos or diagrams of positive and/or negative results.
  • Oligonucleotide hybridization using allele specific probes can be sued to determine the presence or absence of nucleotide sequences that hybridize to PLA2s specific probes.
  • Probes that hybridize to normal PLA2s genes but that will not hybridize to known mutations of PLA2s genes or probes that hybridize to mutated PLA2s genes but that will not hybridize to the normal PLA2s gene can be used to detect the presence or absence of mutations and/or normal genes.
  • Isolated nucleic acid molecules that comprise a nucleotide sequence identical or complementary to a fragment of the PLA2s gene, particularly the coding sequence, that are at least 10 nucleotides are useful to practice the methods of the invention.
  • the isolated nucleic acid molecules consist of a nucleotide sequence identical or complementary to a fragment of the PLA2s gene, particularly the coding sequence, which is at least 10 nucleotides.
  • the isolated nucleic acid molecules comprise or consist of a nucleotide sequence identical or complementary to a fragment of the PLA2s gene, particularly the coding sequence, which is 15-150 nucleotides.
  • the isolated nucleic acid molecules comprise or consist of a nucleotide sequence identical or complementary to a fragment of the PLA2s gene, particularly the coding sequence, which is 15-30 nucleotides.
  • Isolated nucleic acid molecules that comprise or consist of a nucleotide sequence identical or complementary to a fragment of the PLA2s gene, particularly the coding sequence, which is at least 10 nucleotides are useful as probes for identifying PLA2s genes in genomic samples and cDNA sequence generated from RNA from samples, PCR primers for amplifying PLA2s genes and cDNA.
  • the nucleotide sequences in the PLA2s gene particularly the coding sequence be used to design probes, primers and complimentary molecules which specifically hybridize to allele specific PLA2s sequences.
  • the present invention also includes labelled oligonucleotides which are useful as probes for performing oligonucleotide hybridization methods to identify the presence or absence of PLA2s nucleotide sequences. Accordingly, the present invention includes probes that can be labelled and hybridized to allele specific nucleotide sequences of the PLA2s gene, particularly the coding sequence.
  • the labelled probes of the present invention are labelled with radiolabelled nucleotides or are otherwise detectable by readily available nonradioactive detection systems.
  • probes comprise oligonucleotides consisting of between 10 and 100 nucleotides.
  • probes comprise oligonucleotides consisting of between 10 and 50 nucleotides.
  • probes comprise oligonucleotides consisting of between 12 and 20 nucleotides.
  • the probes preferably contain nucleotide sequence completely identical or complementary to a fragment of an allele specific nucleotide sequences.
  • kits for performing oligonucleotide hybridization analysis which comprise an container having a PLA2s specific probe and instructions for performing the assay.
  • positive and/or negative controls may be provided and/or representative photos or diagrams of positive and/or negative results.
  • Genomic PLA2s genes sequences are amplified by PCR and analyzed to detect the presence or absence of sequence mutations. Genomic samples may be obtained from tissue, blood or other body fluids by routine collection methods. Chromosomal DNA is extracted and examined. The presence of mutated PLA2s genes and/or the absence of normal PLA2s genes can be detected.
  • direct sequencing of coding sequence and sequences adjacent to exons is performed following amplification of such sequences.
  • a comparison of the sequence of amplified DNA to the known normal sequence indicates the presence or absence of mutations. Mutations can also be identified using single strand conformation polymorphism (SSCP) and duplex analysis using amplified sequences and constructs having normal sequences.
  • fragment size analysis may be performed using PCR amplified microsatellite DNA. The actual size of amplified microsatellite DNA may be compared to the predicted size of normal microsatellite DNA. Deviation of the actual size of amplified microsatellite DNA from the predicted size of normal microsatellite DNA indicates a mutation in the PLA2s gene.
  • PCR may be used to amplify all or a portion of the genomic sequence that encode Pla2.
  • PCR technology is practiced routinely by those having ordinary skill in the art and its uses in diagnostics are well known and accepted. Methods for practicing PCR technology are disclosed in "PCR Protocols: A Guide to Methods and Applications", Innis, M.A. , et al . Eds. Academic Press, Inc. San Diego, CA (1990) which is incorporated herein by reference. Applications of PCR technology are disclosed in "Polymerase Chain Reaction” Erlich, H.A., et al . , Eds. Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) which is incorporated herein by reference. Some simple rules aid in the design of efficient primers.
  • Typical primers are 18-28 nucleotides in length having 50% to 60% g+c composition.
  • the entire primer is preferably complementary to the sequence it must hybridize to.
  • primers generate PCR products 100 basepairs to 2000 base pairs. However, it is possible to generate products of 50 base pairs to up to 10 kb and more.
  • PCR primers are designed which amplify the region of the PLA2s gene that includes the site where mutations occur.
  • Primers ABl, AB2, AB3 and AB4 amplify the region upstream of the first exon and exon one. This region contains information for regulating gene expression.
  • AB4 TGCTCTTGACAGGACATCAG (SEQ ID NO:9)
  • Primers msatl and msat2 amplify the region surrounding the microsatellite and can be used to detect microsatellite instability.
  • msatl TGTATCATGGGGTTCTCCAC (SEQ ID NO:10)
  • msat2 TCCCATCCAACCTAAGTCCA (SEQ ID NO: 11)
  • Primers ex21 and ex22 amplify exon 2 and the surrounding sequences.
  • ex21 AGTGTGACAGAGGAAGTCAC (SEQ ID NO:12)
  • ex22 TTGGGAGTTGTCTGGTGATG
  • Primers ex31 and ex32 amplify exon 3 and the surrounding sequences.
  • kits for performing genomic DNA analysis which comprise an container having a set PLA2s specific primers such as those described herein and instructions for performing the assay.
  • positive and/or negative controls may be provided and/or representative photos or diagrams of positive and/or negative results.
  • the sequence which hybridizes to the amplified sequences may be provided in a container.
  • the present invention relates to a transgenic non- human mammal that comprises the recombinant expression vector that comprises a nucleic acid sequence that encodes the PLA2s.
  • Transgenic non-human mammals are the endogenous ability to produce function PLA2s. Examples include Black 6 mice which are PLA2s " . The techniques for generating transgenic animals are well known.
  • transgenic animals which express PLA2s.
  • Preferred animals are rodents, particularly goats, rats and mice. Such animals are useful as animal models to identify and characterize PLA2s inhibitors.
  • Parent lines of the mice normally develop multiple polyps. The presence and expression of the transgene reduces the number of polyps which develop. Effective PLA2 inhibitors will inactivate the PLA2s expressed by the transgene and the transgenic mice will revert to the phenotype similar to the parent line.
  • the present invention provides prophylactic methods for preventing or reducing the risk of colon cancer. These methods essentially supply the patient identified as being in a higher risk group an amount of functional PLA2s to compensate for any deficiency.
  • an individual is screened using the identification methodology described above to establish a PLA2s mutation or PLA2s protein or enzyme activity deficiency.
  • the patient is also screened for APC genotype to further characterize the risk group to be assigned.
  • a composition is administered to such an individual to counteract the deficiency and provide the individual with sufficient PLA2s to reduce the risk of colon cancer.
  • the method of the invention thus comprises 1) identifying individuals with PLA2s mutations by genotyping or PLA2s protein or enzyme activity deficiencies by biopsy or stool sample analysis, and 2) administering to such an individual a composition effective to provide the individual with sufficient PLA2s to make up for the PLA2s deficiency. Providing the individual with sufficient PLA2s to make up for the PLA2s deficiency renders the individual less likely to develop polyps, thereby reducing the risk of colon cancer.
  • compositions for delivering PLA2s to individuals identified as being at an elevated risk of colon cancer in order to prevent or reduce the risk of such individuals developing colon cancer.
  • a homozygous mutation in the PLA2s gene indicates the individual has an extremely elevated risk of familial colon cancer.
  • a heterozygous mutation in the PLA2s gene indicates the individual has an elevated risk of familial colon cancer.
  • a homozygous mutation in the PLA2s gene indicates the individual has an elevated risk of spontaneous colon cancer.
  • a heterozygous mutation in the PLA2s gene indicates the individual has a somewhat elevated risk of spontaneous colon cancer.
  • Genotype individuals to detect PLA2s mutations and determine whether individuals are at high risk is useful in the health management of such individuals.
  • Individuals identified as being at genetically higher risk of colon cancer can be monitored extensively and more often than is prescribed for individuals not genetically predisposed to colon cancer.
  • individuals identified as being at higher risk of colon cancer can take preventative steps and treatments to reduce and minimize the risk of colon cancer.
  • compositions include protein compositions, PLA2s genes in combination with the appropriate vectors including gene therapy vectors or microbial vectors.
  • compositions according to the invention comprise a pharmaceutically acceptable carrier in combination with phospholipase, particularly, PLA2s.
  • phospholipase particularly, PLA2s.
  • Pharmaceutical formulations for are well known and pharmaceutical compositions comprising phospholipase may be routinely formulated by one having ordinary skill in the art. Suitable pharmaceutical carriers are described in Remington ' s Pharmaceutical Sciences, A.
  • the present invention relates to an pharmaceutical composition that comprises a pharmaceutically acceptable carrier and phospholipase, particularly, PLA2s.
  • phospholipase is preferably sterile and combined with a sterile pharmaceutical carrier.
  • PLA2s can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable vehicle.
  • a pharmaceutically acceptable vehicle examples include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used.
  • the vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives) .
  • the formulation is sterilized by commonly used techniques.
  • An injectable composition may comprise ICEe and/or ICE ⁇ in a diluting agent such as, for example, sterile water, electrolytes/dextrose, fatty oils of vegetable origin, fatty esters, or polyols, such as propylene glycol and polyethylene glycol.
  • a diluting agent such as, for example, sterile water, electrolytes/dextrose, fatty oils of vegetable origin, fatty esters, or polyols, such as propylene glycol and polyethylene glycol.
  • the injectable must be sterile and free of pyrogens.
  • the phospholipase protein administered orally or rectally. In cases where phospholipase is administered orally, it is provided in an enteric formulation so that it can avoid degradation by stomach acids. Enteric formulations are described in U.S. Patent Number 4,601,896, U.S. Patent Number 4,729,893, U.S. Patent Number 4,849,227, U.S.
  • Patent Number 5,271,961, U.S. Patent Number 5,350,741, and U.S. Patent Number 5,399,347 which are each hereby incorporated herein by reference.
  • Oral and rectal formulation are taught in Remington's Pharmaceutical Sciences, 18th Edition, 1990, Mack Publishing Co., Easton PA. which is incorporated herein by reference.
  • Alternative embodiments include sustained release formulations and implant devices which provide continuous delivery of phospholipase to the colon.
  • compositions according to the invention include delivery components in combination with nucleic acid molecules that encode PLA2s which further comprise a pharmaceutically acceptable carriers or vehicles, such as, for example, saline. Any medium may be used which allows for successful delivery of the nucleic acid.
  • a pharmaceutically acceptable carriers or vehicles such as, for example, saline. Any medium may be used which allows for successful delivery of the nucleic acid.
  • pharmaceutically acceptable media such as, for example, saline.
  • compositions of the present invention may be administered by any means that enables the active agent to reach the agent's site of action in the body of a mammal.
  • Pharmaceutical compositions may be administered parenterally, i.e., intravenous, subcutaneous, intramuscular. Intravenous administration is the preferred route. Dosage varies depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
  • a prophylactically effective dose is one in which the incidence of polyp development or cancer is decreased upon administration of such a dose compared to the incidence of polyp development or cancer which would occur in the absence of such a dose. It is contemplated that a prophylactically effect vie dose will be that which raised the level of functional PLA2s enzyme activity in an individual identified as being in a high risk group to the level of a normal individual without PLA2s mutations or deficiencies.
  • Nucleic acid molecules that encode pla2s may be delivered using any one of a variety of delivery components, such as recombinant viral expression vectors or other suitable delivery means, so as to affect their introduction and expression in the patient's cells, particularly colon cells.
  • viral vectors may be DNA viruses such as recombinant adenoviruses and recombinant vaccinia viruses or RNA viruses such as recombinant retroviruses.
  • Other recombinant vectors include recombinant prokaryotes which can infect cells and express recombinant genes.
  • other delivery components are also contemplated such as encapsulation in liposomes, transferrin- mediated transfection and other receptor-mediated means.
  • the invention is intended to include such other forms of expression vectors and other suitable delivery means which serve equivalent functions and which become known in the art subsequently hereto.
  • DNA is delivered to competent host cells by means of an adenovirus.
  • an adenovirus One skilled in the art would readily understand this technique of delivering DNA to a host cell by such means.
  • the invention preferably includes adenovirus, the invention is intended to include any virus which serves equivalent functions.
  • RNA is delivered to competent host cells by means of a retrovirus.
  • a retrovirus Any retrovirus which serves to express the protein encoded by the RNA is intended to be included in the present invention.
  • nucleic acid is delivered through folate receptor means.
  • the nucleic acid sequence to be delivered to a cell is linked to polylysine and the complex is delivered to cells by means of the folate receptor.
  • the nucleotide sequence that encodes PLA2s is inserted into an expression vector of a non-toxic enteric bacteria host.
  • the host is then administered to an individual where it colonizes the gut and produces PLA2s.
  • suitable host strains include E. coli, Lactobacillus sp. and bacteria describe din Fu, et al . 1994 Cancer Research 54:6297-6301 and Virgilio, et al . 1994 Proc. Natl . Acad. Sci . USA 91:12530-12534 which are both incorporated herein by reference.
  • expression vector is meant to refer to a plasmid, phage, viral particle or other vector which, when introduced into an appropriate host, contains the necessary genetic elements to direct expression of the coding sequence that encodes the PLA2s.
  • the coding sequence is operably linked to the necessary regulatory sequences.
  • Expression vectors are well known and readily available. Examples of expression vectors include plasmids, phages, viral vectors and other nucleic acid molecules or nucleic acid molecule containing vehicles useful to transform host cells and facilitate expression of coding sequences.
  • Another aspect of the invention relates to the administration of compounds useful to upregulate expression of endogenous PLA2s in an individual identified as having an APC mutation.
  • Such compounds include TNF, IL-1, IL-6 and endotoxins.
  • Example 1 Blood samples or appropriate tissue samples are taken from patients for analysis. Blood samples can be taken for genotyping. Alternatively, tissue biopsies can be taken from individuals being examined for other maladies.
  • Genomic DNA is isolated from these samples by standard procedures. The DNA is analyzed for PLA2s allele/polymorphism/mutation. Several protocols exist to determine status of PLA2s allele. The most direct protocol is to amplify the entire gene with the specified oligonucleotides Oligo pair Size amplified and sequenced
  • the PCR conditions are as follows: After PCR the fragments are purified on a Quagen column and sequenced by TAQ DYE-DEOXY sequencing an automated PCR method based on Sanger-Coulson method of DNA sequencing. The results are analyzed and compared with known PLA2s from both human, mouse and rat genes.
  • sequence data is examined for gross alternations such as large insertions or deletions of nucleotide sequences, especially in the open reading frame. Sequence data is also examined to identify single base insertion and deletions.
  • Min mice Multiple intestinal neoplasia mice carry a dominant mutation in the murine homolog of the Ape (Adenomatous polyposis coli) gene. Min mice develop multiple adenomas throughout their small and large intestine. Recent studies have identified a modifier locus, Moml, which maps to the distal region of chromosome 4. Moml dramatically influences Min-induced tumor number. We report here the identification of a candidate gene for Moml.
  • PLA2s The gene for secretory type II Phospholipase A2 maps between D4Mitl6 and D4Mitl3 on the distal region of chromosome 4, the same region that contains Moml, and displays 100% concordance between allele type and tumor susceptibility.
  • Expression and sequencing analysis revealed that Moml susceptible strains are most likely null for PLA2s activity.
  • Our results indicate that PLA2s acts as a novel gene which modifies polyp number by altering the cellular microenvironment within the intestinal crypt.
  • the Min (Multiple mtestinal neoplasia) strain established from an ethylnitrosourea-treated C57BL/6J (B6) male mouse, carries a fully penetrant dominant mutation. Mice heterozygous for the Min mutation develop numerous adenomas throughout the small and large intestinal tract and severe anemia (a result of intestinal bleeding) . The observed phenotype in the Min animals closely resembles the clinical features observed in patients with FAP. Genetic linkage analysis localized the Min mutation to mouse chromosome 18, in a region known to contain the Ape gene, the murine homolog of the human APC gene. Further studies revealed that the Min mutation (hereafter called Apc Min ) results from a nonsense mutation in exon 15 of the Ape gene; the same type of mutation is frequently found in human FAP kindreds.
  • Apc Min the Min mutation results from a nonsense mutation in exon 15 of the Ape gene; the same type of mutation is frequently found in human FAP kindreds.
  • Min mice differ greatly in the number of intestinal tumors depending on their genetic background.
  • B6 mice heterozygous for the Apc Min mutation exhibit an average of 29 tumors throughout their small and large intestines, while their FI progeny produced from crosses to either AKR/J (AKR) , MA/MyJ or Mus castaneus (CAST/Ei) mice exhibit an average of only 5.8, 5.7, and 3.0 tumors, respectively.
  • QTL Quantitative trait loci mapping subsequently identified an unlinked locus, Moml (Modifier of Min 1) , in the distal region of chromosome 4 between D4Mitl6 and D4Mitl3.
  • Moml is estimated to account for approximately 50% of the genetic variation in adenoma number in both AKR and MA/MyJ intraspecific backcrosses as compared to B6. Interestingly, Moml also resides in a region of synteny with human chromosome lp35, a region frequently associated with loss of heterozygosity in human plasmacytomas, neuroblastomas and colon cancer. It has not yet been determined whether the product of the Moml gene product acts autonomously or non-cell autonomously with respect to the tumor cell lineage. PCR analysis has shown that 100% of spontaneous Min-induced intestinal adenomas have lost their wildtype Ape allele, suggesting that Ape acts as a classical tumor suppressor gene.
  • Multilabel immunocytochemical analysis of these adenomas has demonstrated that the intestinal lesions consist of a mixture of differentiated enterocytes, enteroendocrine, goblet and Paneth cells; these findings are similar to what is observed in human adenomatous polyps.
  • the presence of multiple lineages in these adenomas suggests that a multipotent stem cell population located in the base of intestinal crypts is the site of initiation for Apc Min induced tumorigenesis. Therefore, any modifier gene expressed in the crypt microenvironment would have a potential role in altering tumor progression.
  • PLA2s is the first genetically defined locus that can modify or influence the number of intestinal tumors resulting from a mutation in the Ape gene.
  • Results Chromosomal location of the PLA2s gene in the mouse An interspecific backcross mapping panel was used to localize PLA2s in the mouse. Genomic DNAs from AEJ/Gn and Mus spretus parental control mice were digested with fourteen restriction endonucleases and analyzed by Southern blot hybridization using the PLA2s probe. An informative restriction fragment length polymorphism (RFLP) was detected with the restriction endonuclease BamHI (see Experimental Procedures) .
  • RFLP restriction fragment length polymorphism
  • the segregation pattern of the PLA2s gene was followed in 195 N2 progeny; each mouse was either homozygous for the AEJ/Gn allele or heterozygous for the M. spretus and AEJ/Gn alleles.
  • the allele distribution pattern of PLA2s was compared to known gene and microsatellite markers which scan the entire mouse genome.
  • the segregation analysis revealed that the PLA2s gene resides on mouse chromosome 4.
  • the specific location of PLA2s was determined by minimizing the number of multiple recombinations along the length of the chromosome ( Figure 1 Panel A) .
  • the results positioned PLA2s between D4Mitl48 and D4Mitl3 ( Figure 1 Panel B in the distal region of chromosome 4.
  • the order of the loci and the ratio of the number of recombinants to the total number of N2 offspring examined are: centromere - D4Mitl6 - 19/183 - D4Mitl48 - 1/170
  • PLA2s we hypothesized that if PLA2s was involved, the level or quality of mRNA or protein expression would differ between susceptible (Moml s ) and resistant (Moml r ) strains.
  • PLA2s expression was limited to the small and large intestine of the AKR strain, where an 800 bp transcript was detectable after an overnight exposure to autoradiography film ( Figure 2) . In contrast, no transcript was detected in the small or large intestine of B6 mice after a similar exposure.
  • PLA2s expression in MA/MyJ and CAST/Ei two strains also determined to contain the Moml r allele. High levels of the 800 bp transcript were detected in the small and large intestines of these strains ( Figure 2 Panel A) .
  • Northern blot analysis identified 2 strains, C3H/HeJ and CBA/J, with high levels of
  • PLA2s expression in the large and small intestine However, northern blot analysis revealed 4 strains, P/J, A/J, C58/J
  • PLA2s similar to the expression level observed in B6 mice. Though none of these strains have been tested for their Moml phenotype, we predict that the C3H/HeJ and CBA/J strains carry a Moml r allele, while the A/J, P/J, C58/J and 129/SvJ strains carry a Moml s allele. Consistent with this prediction, it has recently been reported that 129/SvJ mice carrying a targeted mutation in the Ape gene develop high numbers of intestinal adenomas, indicating that the 129/SvJ strain carries a susceptible Moml allele.
  • the low levels of PLA2s expression observed in B6 mice could be the result of a mutation in the promoter region resulting in a decreased transcription rate or a mutation in the transcribed region resulting in a decreased half-life of the PLA2s mRNA.
  • genomic DNA from B6 and AKR mice was digested with several restriction endonucleases to identify RFLPs for the PLA2s gene.
  • Southern blot analysis identified three enzymes, BamHI, Mspl and TaqI that revealed RFLPs between the B6 and AKR strains.
  • the mouse PLA2s cDNA was isolated from an ileal cDNA (C57BL/6J X C2H)FI library. Screening with a rat PLA2s cDNA probe yielded two positive clones, MPla2s-2 and MPla2s-6. Both clones were most likely derived from the C3H allele, since PLA2s expression is much higher in C3H mice than in B6 mice. Sequence analysis revealed that both clones contained the entire PLA2s open reading frame including the N-terminal signal peptide ( Figure 4) .
  • Clone MPla2s-6 also included a potential polyadenylation site closely followed by a stretch of adenines indicative of poly-A+ tail. Sequence comparison indicated that the clones were 99% identical at the nucleotide level with the available murine PLA2s sequence determined from BALB/c cDNA (GENBANK Accession #X74266) and 86% identical at the nucleotide level with the rat PLA2s sequence.
  • oligodeoxynucleotide primers derived from the 5' and 3' noncoding regions were prepared based on the sequence of the MPla2s-6 cDNA clone (see Experimental Procedures) . All the strains assayed, regardless of the PLA2s allele, produced a PCR product of approximately 720 bp. In addition to the 720 bp product, all the inbred strains containing the 9.0 kb BamHI allele produced a smaller 610 bp PCR product ( Figure 5 Panel A) .
  • the mutated sequence 5' -GGATTCC-3' (SEQ ID NO:20) is detected in all strains containing the 9.0 kb BamHI allele and exhibiting reduced expression of PLA2s by northern blot analysis.
  • the insertion results in a frameshift mutation that creates a stop codon 13 amino acids downstream in exon 4 ( Figure 4) .
  • the RT-PCR product from the AKR strain contained the BamHI site that was observed in both C3H/HeJ and BALB/c mice ( Figure 4) .
  • the BamHI site was detected in all inbred strains possessing the 2.5 and 6.5 kb BamHI restriction, consistent with this site causing the polymorphism.
  • PLA2s is one of several enzymes involved in generating arachidonic acid and lysophosphatidic acid.
  • Arachidonic acid is the rate limiting substrate for the generation of prostaglandins (via the cyclooxygenase pathway) and leukotrienes (via the lipoxygenase pathway) .
  • Phospholipase C and prostaglandins have been found. The role of prostaglandins in the susceptibility to adenoma formation is unclear.
  • NSAIDs non- steroidal anti-inflammatory drugs
  • the mechanism of action of the NSAIDs, such as Sulindac is presumed to be by inhibiting cyclooxygenase, resulting in a decrease in prostaglandin production.
  • various investigators hypothesized that the decrease of specific prostaglandins contributes to tumor protection and regression.
  • PLA2s might interact with the intestinal villi to stimulate the formation of aberrant crypts. Therefore, the presence of high levels of PLA2s in the intestine would provide a protective effect by inactivating the harmful affects of dietary fatty acids.
  • PLA2s might be important at maintaining normal intestinal flora; different types of dietary fat have been associated with alteration of the normal intestinal flora. Diets high in saturated fatty acid are associated with increased amounts of the anaerobic bacteria, Bacteroids. It has recently been demonstrated that purified type II PLA2s, isolated from mouse intestines, possesses potent bactericidal activity.
  • PLA2s is secreted from Paneth cells and these cells secrete many bactericidal proteins, including lysozyme and defensins it is not surprising that PLA2s has a role in microbial defense mechanisms.
  • the Moml r strains have high levels of PLA2s in their intestinal lumen to help protect and/or control the intestinal flora, while the Moml s strains would lack sufficient levels of PLA2s to affect bactericidal activity.
  • Bacteroides and other anaerobic bacteria have been proposed to produce toxins or convert bile salts to carcinogenic products.
  • one potential mechanism of increased adenoma susceptibility in B6 mice is that the lack of PLA2s in the intestine allows for the proliferation of certain types of bacteria that produce carcinogenic products which facilitate polyp formation and transformation.
  • Murine intestinal PLA2s was independently identified as Enhancing Factor. Enhancing Factor was identified as a low molecular weight heat and acid stable polypeptide that enhanced the binding of EGF to cells. In addition, several reports have identified a mitogenic action for PLA2s. PLA2s was found to induce DNA synthesis and proliferation independent of arachidonate products. Thus, the presence of high levels of PLA2s in intestinal crypts might provide an environment that would inhibit the formation of transformed crypts due to the enhanced accessibility of growth factors.
  • PLA2s activity requires millimolar concentrations of Ca + , suggesting that PLA2s must be released into the extracellular environment to become fully active.
  • specificity of PLA2s in the microenvironment is mediated by membrane asymmetry; specifically, PLA2s has a preference for the head group of anionic phospholipids (i.e. phosphatidylserine (PS) and phosphatidylethanolamine (PE) located on the external membrane.
  • PS phosphatidylserine
  • PE phosphatidylethanolamine
  • Normal colonic epithelium has phosphatidylcholine (PC) on its external leaflet, the phospholipid which is a low affinity substrate of PLA2s.
  • PLA2s does not catalytically attack the membrane phospholipids of normal intestinal epithelium.
  • bacterial cell membranes comprised mostly of phosphatidylglycerol (PG)
  • PG phosphatidylglycerol
  • Colon carcinoma cells have been shown to have elevated levels of PS on their outer leaflet, making a suitable target for PLA2s digestion.
  • PLA2s represents a new class of genes that influence tumor susceptibility. We have proposed several mechanisms to explain how PLA2s activity could contribute to tumor resistance. Regardless of the mechanism, it is probable that the mode of protection is non-cell autonomous, since PLA2s is most likely active in the intestinal lumen and not within the cell. Recently, another modifier of murine intestinal cancer that is intrinsic to the transformed cell has been identified. This modifier confers tumor resistance by causing DNA hypomethylation. The identification of this cell autonomous modifier was accomplished through the use of gene targeting and drug treatment in mice.
  • the proposed models for PLA2s action are also testable through the use of mouse mutants and through experimental manipulation of the diet and drug treatment of Moml s and Moml r strains.
  • the ultimate proof of PLA2s action as a modifier of tumor multiplicity in intestinal cancer will be shown by expressing a wildtype copy of the PLA2s gene within the microenvironment of the intestinal crypts in Moml s strains and demonstrating a reduction in the number of adenomas or by targeted ablation of the PLA2s gene in Moml r strains.
  • the human homolog of PLA2s has been localized to chromosome lp35, a region that exhibits loss of heterozygosity in a variety of neoplasms.
  • chromosome lp35 a region that exhibits loss of heterozygosity in a variety of neoplasms.
  • the identification of PLA2s as the genetically defined Moml locus has several important implications in human cancer.
  • One possibility of PLA2s involvement may be reflected by the variable number of adenomas identified amongst family members inheriting the same APC mutation. his variation in tumor burden could be attributable to the independent segregation of different PLA2s alleles that confer variations in expression levels or enzyme activity.
  • allelic variation at the PLA2s locus could contribute to the susceptibility of non- familial colon cancer within the general population.
  • population surveys would be useful to determine the extent of allelic variation as well as to provide a potential predictor to identify individuals at risk for developing intestinal cancer.
  • identification of PLA2s as a major modifier of intestinal polyp formation may provide a missing link between high fat diets and increased incidence of colon cancer.
  • mice The murine chromosomal location of PLA2s was determined using interspecific backcross analysis.
  • the interspecific backcross was [(AEJ/Gn - a bp H /a bp H x Mus spretus)F x X AEJ/Gn - a bp H ] .
  • Additional inbred strains were purchased from The Jackson Laboratory (Bar Harbor, ME) . Probes
  • Probes used to map the mouse PLA2s gene and screen murine cDNA libraries were derived from a 758 bp rat cDNA fragment cloned into the EcoRI site of a pGEM vector.
  • the pGEM vector containing the rate PLA2s gene was a gift from Dr. J. Ishizaki (Shionogi Research Laboratories, Osaka, Japan) .
  • the chromosomal location of PLA2s was determined by Southern blot analysis in 195 N2 animals.
  • a 456 bp fragment of the rat PLA2s coding region was amplified by the PCR using the following oligodeoxynucleotide primers (ZW-1) 5' - ATGAAGGTCCTCCTGTTG-3' SEQ ID NO:21 and (ZW-2) 5'-CAGAGAGTGTCTTTTCAGC-3' SEQ ID NO:22. These primers correspond to bases 1 and 456 of the rat PLA2s coding region (GENBANK Accession #M37127) .
  • the resulting PCR fragment was radiolabeled with [a- 32 P] -dCTP using a random primed kit (Boehringer Mannheim, Indianapolis, IN) and used for Southern blot analyses.
  • cDNA cloning Murine PLA2s clones (MPLA2sl-6) were isolated from a (C57BL/6J x C3H)F1 ileal cDNA library made from the terminal 3.0 cm of the small intestine; the library was a generous gift from Dr. J. Gordon (Washington University School of Medicine, St. Louis, MO) . The clones were isolated following standard techniques. A 780 bp fragment of the entire coding region was amplified as above, using T3 and T7 primers which flank the insert. PCR products were purified by electrophoresis on a Seaplaque low-melt agarose gel, radiolabeled and used for northern and Southern blot analysis. SSLP Analysis
  • DNA primers for SSLP analyses were made using an Applied Biosystems Model 394 DNA/RNA synthesizer. Genomic DNA isolation and agarose gel electrophoresis were performed. Primers and fragment sizes for the D4Mitl3, D4Mitl6 and D4Mitl48 microsatellite markers were as described in Dietrich et al., 1992 Genetics 131:423-447 which is incorporated herein by reference) . PCR conditions were performed as described (Ma et al., 1993 Proc. Natl. Acad. Sci. USA 90:6350-6354).
  • the PCR protocol for the primes was an initial denaturation at 94 C for 4 min, followed by 40 cycles at 94 C for 30 sec, 55-60 C for 30 sec, 72 C for 30 sec, and ended with one 72 C extension for 7 min.
  • the resulting D4Mitl48 fragments were visualized by ethidium bromide staining Of 3% agarose gels, while 10% and 5% polyacrylamide gels were used for D4Mitl3 and D4Mitl6, respectively.
  • DNA isolation and Southern Blot Analysis Genomic DNAs from a variety of inbred strains were isolated from tail biopsies.
  • RNA Isolation and Northern Blot Analysis Total RNA from mouse tissues was isolated. 20 g of total RNA was size-fractionated on 1.0% formaldehyde agarose gels. Northern blots were transferred and hybridized. RT-PCR and Sequence Analysis
  • First strand cDNA was synthesized from 1.0 g of total RNA using oligo d(T) 15 and M-MuLV reverse transcriptase. PCR was performed, as described above, using oligodeoxynucleotide primers specific for murine PLA2s. Double stranded cDNA products were purified using QIAquick PCR purification columns
  • Oligodeoxynucleotide primers for PCR analysis were synthesized with -cyanoethyl phophoramidities on an Applied Biosystems 394 DNA/RNA synthesizer.
  • immunoassay comprise allowing proteins in the sample to bind a solid phase support such as a plastic surface. Detectable antibodies are then added which selectively binding to PLA2s. Detection of the detectable antibody indicates the presence of PLA2s.
  • the detectable antibody may be a labelled or an unlabelled antibody. Unlabelled antibody may be detected using a second, labelled antibody that specifically binds to the first antibody or a second, unlabelled antibody which can be detected using labelled protein A, a protein that complexes with antibodies.
  • labelled protein A a protein that complexes with antibodies.
  • Various immunoassay procedures are described in Immunoassay for the 80 's, Voller, et al . , Ed., University Park, 1981, which is incorporated herein by reference. The amount of antibodies present may be quantified by well known techniques.
  • Simple immunoassay may be performed in which a solid phase support is contacted with the test sample. Any proteins present in the test sample bind the solid phase support and can be detected by a specific, detectable antibody preparation. Such a technique is the essence of the dot blot, Western blot and other such similar assays. Other immunoassay may be more complicated but actually provide excellent results.
  • Typical and preferred immunometric assays include "forward" assays for the detection of a protein in which a first anti-protein antibody bound to a solid phase support is contacted with the test sample. After a suitable incubation period, the solid phase support is washed to remove unbound protein. A second, distinct anti-protein antibody is then added which is specific for a portion of the specific protein not recognized by the first antibody.
  • the second antibody is preferably detectable. After a second incubation period to permit the detectable antibody to complex with the specific protein bound to the solid phase support through the first antibody, the solid phase support is washed a second time to remove the unbound detectable antibody. Alternatively, the second antibody may not be detectable. In this case, a third detectable antibody, which binds the second antibody is added to the system.
  • This type of "forward sandwich” assay may be a simple yes/no assay to determine whether binding has occurred or may be made quantitative by comparing the amount of detectable antibody with that obtained in a control.
  • Such "two-site” or “sandwich” assays are described by Wide, Radioimmune Assay Method, (1970) Kirkham, Ed., E. & S. Livingstone, Edinburgh, pp. 199-206, which is incorporated herein by reference.
  • a simultaneous assay involves a single incubation step wherein the first antibody bound to the solid phase support, the second, detectable antibody and the test sample are added at the same time. After the incubation is completed, the solid phase support is washed to remove unbound proteins. The presence of detectable antibody associated with the solid support is then determined as it would be in a conventional "forward sandwich” assay.
  • the simultaneous assay may also be adapted in a similar manner for the detection of antibodies in a test sample.
  • the "reverse” assay comprises the stepwise addition of a solution of detectable antibody to the test sample followed by an incubation period and the addition of antibody bound to a solid phase support after an additional incubation period.
  • the solid phase support is washed in conventional fashion to remove unbound protein/antibody complexes and unreacted detectable antibody.
  • the determination of detectable antibody associated with the solid phase support is then determined as in the "simultaneous" and "forward" assays.
  • the reverse assay may also be adapted in a similar manner for the detection of antibodies in a test sample.
  • the first component of the immunometric assay may be added to nitrocellulose or other solid phase support which is capable of immobilizing proteins.
  • the first component for determining the presence of PLA2s in a test sample is anti- PLA2s antibody.
  • solid phase support or “support” is intended any material capable of binding proteins.
  • Well-known solid phase supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the support can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc.
  • suitable solid phase supports for binding proteins or will be able to ascertain the same by use of routine experimentation.
  • a preferred solid phase support is a 96-well microtiter plate.
  • detectable anti-PLA2s antibodies are used. Several methods are well known for the detection of antibodies. Anti-PLA2s antibodies may be labelled with a radioisotope and the amount of radioisotope may be determined using a scintillation counter.
  • One method in which the antibodies can be detectably labelled is by linking the antibodies to an enzyme and subsequently using the antibodies in an enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) , such as a capture ELISA.
  • EIA enzyme immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the enzyme when subsequently exposed to its substrate, reacts with the substrate and generates a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means.
  • Enzymes which can be used to detectably label antibodies include, but.
  • malate dehydrogenase staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • yeast alcohol dehydrogenase alpha-glycerophosphate dehydrogenase
  • triose phosphate isomerase horseradish peroxidase
  • alkaline phosphatase asparaginase
  • glucose oxidase beta-galactosidase
  • ribonuclease urease
  • catalase glucose-6-phosphate dehydrogenas
  • radioactive isotopes Another method in which antibodies can be detectably labelled is through radioactive isotopes and subsequent use in a radioimmunoassay (RIA) (see, for example, Work, et al . , Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, N.Y., 1978, which is incorporated herein by reference) .
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are 3 H, 15 I, 131 I, 35 S, and 14 C. Preferably 125 I is the isotope.
  • 125 I is the isotope.
  • One skilled in the art would readily recognize other radioisotopes which may also be used.
  • fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • fluorescein isothiocyanate rhodamine
  • phycoerythrin phycocyanin
  • allophycocyanin o-phthaldehyde and fluorescamine.
  • Antibodies can also be detectably labelled using fluorescence-emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the protein-specific antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediamine- tetraacetic acid (EDTA) .
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediamine- tetraacetic acid
  • Antibodies can also be detectably labelled by coupling to a chemiluminescent compound.
  • the presence of the chemiluminescent-labelled antibody is determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label antibodies.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin. One skilled in the art would readily recognize other bioluminescent compounds which may also be used.
  • Detection of the protein-specific antibody, fragment or derivative may be accomplished by a scintillation counter if, for example, the detectable label is a radioactive gamma emitter.
  • detection may be accomplished by a fluorometer if, for example, the label is a fluorescent material.
  • the detection can be accomplished by colorometric methods which employ a substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • One skilled in the art would readily recognize other appropriate methods of detection which may also be used.
  • binding activity of a given lot of antibodies may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • Positive and negative controls may be performed in which known amounts of PLA2s and no PLA2s, respectively, are added to assays being performed in parallel with the test assay.
  • One skilled in the art would have the necessary knowledge to perform the appropriate controls.
  • the protocol yields an active enzyme. 1. Wet weight of tissue is obtained from an individual.
  • a tared beaker is used.
  • Dialysis tubing should have molecular weight cut off of less than 12kD since the protein of interest is 14.5kD). Overnight dialysis in a fresh change of sodium acetate is usually required (one change of fluid) .
  • sample for Western blot analysis, we would take equal volumes of sample (acid extract) , add an aliquot of 5X SDS sample buffer to yield a final concentration of IX SDS sample buffer, and then add sufficient ION NaOH (usually one or two ul) to neutralize sample (as evidenced by restoration of blue color of bromophenol blue pH indicator included in SDS sample buffer) . Samples were then boiled 5 min. , centrifuged in a microfuge to remove insoluble materials (16,000 X G for 15 min.), and loaded onto 20% SDS-gels, 4% stacking gel, 0.75 mm thick. Appropriate low molecular weight standards, as well as authentic human recombinant synovial fluid phospholipase A 2 were loaded on the gel.
  • Electrophoresis was in a Bio-Rad minigel apparatus, at 200V for 45-60 min.
  • the proteins were then transferred to either supported nitrocellulose, or PDVF, in a BioRad mini-transfer apparatus at 30V overnight in the cold, followed by 60V for 2 hr.
  • the gels were recovered and stained with coomassie blue to monitor transfer of the proteins, and the nitrocellulose (or PDVF) was stained with Fast Green to visualize and monitor the transfer of the proteins as well as permit marking of the location of the molecular weight standards.
  • the membranes were then washed, and incubated with polyclonal antibody to human recombinant synovial fluid phospholipase A 2 , or in at least one case, to antibody prepared against mouse intestinal phospholipase A 2 .
  • PLA 2 Assay for group II PLA 2 (PLA, ) • adopted from L.A. Marshall et al., J. Lipid Mediators Cell Signalling 10:295-313 (1994) which is incorporated herein by reference. See also: Patriarca et al. , 1972 Phospholipases and phospholipid turnover in Escherichi Coli Spheroplasts. Biochim Biophys Acta 260:593- 600 which is incorporated herein by reference. This protocol be used with purified enzymes, subcellular fractions, or crude homogenates obtained from tissue biopsy or body fluid samples. - 48 -
  • substrate 100 uM [ 3 H]arachidonic acid labeled E. Coli (about 8 nmol lipid phosphorous) .
  • Bacteria may be radiolabeled using published procedures, or purchases directly from DuPont NEN.
  • the upper phase is transferred to tubes containing lm hexane plus 75g activated BioSil A.
  • the tubes are then vortexed thoroughly, and centrifuged @ 15,000 X g for 10 minutes to produce a supernatant which is then pipetted into scintillation vials, and then evaporated under N 2 .
  • One or more of these treatments would permit determination of the amount of enzyme activity due to the group II PLA 2 by subtraction from assay mixtures that were not treated.

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Abstract

L'invention porte sur des méthodes d'identification d'individus présentant un facteur de risque élevé du cancer du colon, qui consistent à détecter une mutation génique de PLA2s, l'absence de protéine de PLA2s ou l'activité de l'enzyme de PLA2s dans un échantillon prélevé sur un individu. L'invention se rapporte également à des méthodes de prévention du cancer du colon ou de réduction du risque de cancer du colon chez un individu reconnu comme présentant un risque de cancer du colon élevé. Lesdites méthodes consistent à identifier un individu prédisposé au cancer du colon, par détection d'une mutation génique de PLA2s ou d'une activité enzymatique ou protéique déficiente de PLA2s; et à administrer audit individu une dose efficace sur le plan prophylactique de phospholipase ou d'un vecteur de recombinaison constituant une séquence nucléotidique codant PLA2s.
PCT/US1996/009009 1995-06-07 1996-06-06 Detection du facteur de risque du cancer colorectal et traitement WO1996041003A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999024587A3 (fr) * 1997-11-07 1999-07-22 Incyte Pharma Inc Proteine de type phospholipase a2 d'origine humaine et adn la codant
EP1107798A1 (fr) * 1998-08-04 2001-06-20 Diadexus LLC Nouveau procede de diagnostic, de surveillance, de classification par stades, d'imagerie et de traitement du cancer du colon
EP3983431A4 (fr) * 2019-06-17 2023-12-13 The Board of Trustees of the Leland Stanford Junior University Diagnostics et traitements basés sur une caractérisation moléculaire du cancer colorectal

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BRITISH JOURNAL OF CANCER, July 1993, Vol. 68, MURATA et al., "Expression of Group-II Phospholipase A2 in Malignant and Non-Malignant Human Gastric Mucosa", pages 103-111. *
CANCER RESEARCH, 01 March 1996, Vol. 56, SPIRIO et al., "Three Secretory Phospholipase A2 Genes that Map to Human Chromosome 1P35-36 are not Mutated in Individuals with Attenuated Adenomatous Polyposis Coli", pages 955-958. *
CANCER RESEARCH, 15 November 1995, Vol. 55, RIGGINS et al., "Absence of Secretory Phospholipase A2 Gene Alterations in Human Colorectal Cancer", pages 5184-5186. *
CLINICA CHIMICA ACTA, August 1994, Vol. 228, YAMASHITA et al., "Elevation of Serum Group II Phospholipase A2 Levels in Patients with Advanced Cancer", pages 91-99. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999024587A3 (fr) * 1997-11-07 1999-07-22 Incyte Pharma Inc Proteine de type phospholipase a2 d'origine humaine et adn la codant
US6103469A (en) * 1997-11-07 2000-08-15 Incyte Pharmaceuticals, Inc. Human phospholipase A2 protein
US6399301B1 (en) 1997-11-07 2002-06-04 Incyte Genomics, Inc. Human phospholipase A2 protein
EP1107798A1 (fr) * 1998-08-04 2001-06-20 Diadexus LLC Nouveau procede de diagnostic, de surveillance, de classification par stades, d'imagerie et de traitement du cancer du colon
EP1107798A4 (fr) * 1998-08-04 2002-01-09 Diadexus Inc Nouveau procede de diagnostic, de surveillance, de classification par stades, d'imagerie et de traitement du cancer du colon
EP3983431A4 (fr) * 2019-06-17 2023-12-13 The Board of Trustees of the Leland Stanford Junior University Diagnostics et traitements basés sur une caractérisation moléculaire du cancer colorectal

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