CA2070995A1 - Diagnostic probe for detecting human stomach cancer - Google Patents
Diagnostic probe for detecting human stomach cancerInfo
- Publication number
- CA2070995A1 CA2070995A1 CA002070995A CA2070995A CA2070995A1 CA 2070995 A1 CA2070995 A1 CA 2070995A1 CA 002070995 A CA002070995 A CA 002070995A CA 2070995 A CA2070995 A CA 2070995A CA 2070995 A1 CA2070995 A1 CA 2070995A1
- Authority
- CA
- Canada
- Prior art keywords
- met gene
- gene product
- met
- antibody
- human stomach
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 239000000523 sample Substances 0.000 title claims abstract description 14
- 206010017758 gastric cancer Diseases 0.000 title claims abstract description 8
- 208000005718 Stomach Neoplasms Diseases 0.000 title claims abstract description 6
- 201000011549 stomach cancer Diseases 0.000 title claims abstract description 6
- 101150105382 MET gene Proteins 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 12
- 239000012472 biological sample Substances 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 3
- 238000009007 Diagnostic Kit Methods 0.000 claims description 2
- 230000002018 overexpression Effects 0.000 claims 2
- 206010061968 Gastric neoplasm Diseases 0.000 claims 1
- 230000008105 immune reaction Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 10
- 235000018102 proteins Nutrition 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 239000000047 product Substances 0.000 description 6
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 208000009956 adenocarcinoma Diseases 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- DCWXELXMIBXGTH-UHFFFAOYSA-N phosphotyrosine Chemical compound OC(=O)C(N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-UHFFFAOYSA-N 0.000 description 3
- 241000972773 Aulopiformes Species 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 2
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 208000010749 gastric carcinoma Diseases 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 235000019515 salmon Nutrition 0.000 description 2
- 201000000498 stomach carcinoma Diseases 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 102100022623 Hepatocyte growth factor receptor Human genes 0.000 description 1
- 101000972946 Homo sapiens Hepatocyte growth factor receptor Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 208000003252 Signet Ring Cell Carcinoma Diseases 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000011544 gradient gel Substances 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 239000012133 immunoprecipitate Substances 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000001466 metabolic labeling Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 201000008123 signet ring cell adenocarcinoma Diseases 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
Classifications
-
- 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
- G01N33/57446—Specifically defined cancers of stomach or intestine
-
- 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
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/193—Colony stimulating factors [CSF]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/82—Translation products from oncogenes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- 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
- C12Q1/6813—Hybridisation assays
-
- 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
- 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
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- 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
- C12Q2600/158—Expression markers
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- Organic Chemistry (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Oncology (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Hematology (AREA)
- Gastroenterology & Hepatology (AREA)
- Hospice & Palliative Care (AREA)
- Pharmacology & Pharmacy (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
A diagnostic probe for detecting human stomach cancer is described.
Description
2~7~
~ W O 91/09974 . - PC~r/US90/07313 .
DIAGNOSTIC PROBE FOR DETECTING HUMAN STOMACH CANCER
A number of oncogene and proto-oncogene probes have been reported to be useful in diagnostic procedures for detecting certain forms of cancer and for following disease prognosis. However, a specific probe for the diagnosis of human stomach cancer has not heretofore been known or described.
It is, therefore, an object of the present invention to provide specific probes for detecting human stomach cancer. Such a unique probe came into being by the unexpected finding that the met proto-oncogene was activated in certain human cancer cell lines tested. The characteristics of the met gene can be found described in Park et al, 1986, Cell, 45:895-904; Park et al, 1987, PNAS, 84:6379-6383 and Gonzatti-Haces et al, 1988, PNAS, 85:21-25.
- Materials & Methods Various methodologies used for testing the activation of met gene are now described.
Southern Blottinq Genomic DNA from the HOS line and several gastric carcinoma cell lines were digested with EcoRI, ~5 fractionated on a 1% agarose gel, and transferred to nitrocellulose. The blot was baked for 2 hours. The blot was prehybridized for 4 hours at 42-C. The prehybridization and hybridization was in 50~
formamide, 5x Denhardts, 200 ~g/ml salmon sperm DNA, 5x SSPE, and 0.1% SDS. The blot was washed with 2x SSC, 0.1% SD5 for 20 minutes at RT, then with 3 washes of 0.2 x SSC, 0.1% SDS at 68-C.
.
.i - SUBSTITUTE SHEET
.,. . ~ .............. . . .
.
. . .
. ~ .
W O 91/09974 2 ~ ~ O ~ ~ ~ P~r/US90/07313 ~
Northern Blottina Total RNA was fractionated on a 1.1% formaldehyde-agarose gel and transferred to nitrocellulose. The nitrocellulose was baked for 2 hours, then washed 20 minutes in 5x SCC. The blot was prehybridized for 4 hours at 42C. Prehybridization and hybridization w~re in 50% formamide, 0.1% SDS, 6x SSPE, lx Denhardts, and 200 ~g/ml sonicated salmon sperm DNA. The blot was washed 2 times for 10 minutes in 2x SCC, 0.1% SDS at room temperature (about 22~-24C), then 3 times for 20 minutes each in 0.2x SSC, 0.1% SDS at 55C.
35S Metabolic Labeling and Immunoprecipitation Cells were preincubated 15 minutes in DMEM lacking methionine and cysteine supplemented with 10% calf serum, then labeled for 30 minutes in the above media supplemented with 2.5 mCi/ml 35S-methionine and cysteine. Cells were lysed and met was then immunoprecipitated with a mon~clonal directed against the kinase domain. Immunoprecipitates were fractionated on a 3-17% gradient gel. Proteins were detected by fluorography.
32p Metabolic Labelinq Cells were preincubated for 15 minutes with DMEM
lacking phosphate, then labeled for 2 hours in the above media supplemented with 0.5 mCi/ml 32p_ orthophosphate.
Monoclonal Antibody to the met Oncoaene Product A deposit of the hybridoma producing monoclonal antibody having specificity to the met oncogene product has been made at the ATCC, Rockville, MD., on December 14, 1989 under accession number HB 10309. The deposit shall be viably maintained, replacing if it becomes ,~ .
.. :
8VBSTITUT~ SHE~ F-r - . . . . .... . .- .. - . ::
. .
20~9~
~WO91/09974 PCT/US90/07313 non-viable during the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and upon issuance of the patent made available to the public without restriction in accordance with the provision of the law. The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
RESULTS
cFigure 1 shows the gene copy number in gastric carcinoma cell lines as determined by Southern blot. A
fragment representing most of the met cDNA was used to probe genomic DNA which had been digested with EcoRI.
Several hybridizing bands are detected. The MKN-45 and Okajima cell lines show siignificant amplification of the met gene. No rearrangement is detected with this probe. To further confirm these results, RNA levels were examined by Northern blot. Total RNA from several cell lines was probed with a fragment containing the met extracellular domain. A 9 kb met RNA is detected in all cell lines. As shown in Fig. 2, the met RNA is ~
greatly overexpressed in the MXN-45 and Okajima cell lines.
:......................................................................... , Then the expression and half-life of the met protein in some of these lines was examined by pulse chase. Cells were labeled with 35S for thirty minutes and then chased with cold media for l/2, 2 or 4 hours.
~/As shown in Fig. 3, very high levels of the met protein are detected in the MXN-45 line. The protein appears to be processed normally. The MXN-74 and KATOIII lines shown normal protein levels. The Okajima line shows the greatest amount of protein after a 30 minute ~, .
-, 1 SlJBSTlT~lTE SHEE r i - . ~ ..
- , ,- .
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. . - . : ~ . - :
WO9~/09974 2 ~ ~ O 9 9 ~ PCT/US90/07313 ~
labeling, indicating a high rate of synthesis. While the met protein in this line is processed to the 140 form, the 140 is almost all gone after a four-hour chase, indicating a very short half-life.
It was then examined whether the proteins might be phosphorylated on tyrosine. Cells were labeled for 2 hours with 32p orthophosphate and protein was precipitated with an antibody directed against phosphotyrosine. As seen in Fig. 4, both the MKN-45 and Okajima lines show phosphotyrosine present on the pl40 form. No phosphotyrosine was detected on the pl40 in the other cell lines, even with a long overexposure.
Table l summarizes the data presented in Figs. 1-4.
Of the 7 lines examined, two (MKN-45 and Okajima) showed ampIification and phosphorylation on tyrosine on pl40. The Okajima line was unique in displaying a very ~;short half-life for pl40. When these lines were examined for their ability to grow in serum-free media, only the Okajima line was found competent to grow. It ~;20 is significant to note that met was found amplified only in poorly differentiated adenocarcinomas. The other lines examined for which a classification is known included two well differentiated adenocarcinomas ;and signet ring carcinomas.
The results indicate that the met gene is amplified and overexpressed only in poorly differentiated gastric carGinoma cell lines tested. The rapid turnover seen in one line may indicate that an autocrine loop is involved in the genesis of the tumor. Since the met amplification was seen only in poorly differentiated adenocarcinomas, clearly a met probe may be of great value in characterizing the clinical stage to which a ( :
SU E~STITUTE SH ~E~ .
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-. . . . , . - . ,` - . ~ ~ :.- - .:
. .- - :
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~ 091/09974 2 ~ 7 0 9 9 ~ PCT/US90/07313 tumor has progressed. The availability of the monoclonal antibodies to the met gene product now makes ~-it possible not only to isolate and purify, but also to detect the presence of the met gene products in a biological sample by standard i~munological techniques including in situ immunofluorescence or other standard techniques. Accordingly, a diagnostic kit for the detection of met gene product, comprises a container containing antibodies to the met gene product.
lOOf course, given the nucleotide and amino acid sequences, a nucleic acid or polypeptide probe for detecting the met gene or the met gene product is easily made by conventional methodologies well known to one of ordinary skill in the art. Nucleic acid probes useful for this purpose re described in Park et al, supra, while probes are also described in Gonzatti-Haces et al, supra.
It is noted that unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
;Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials have been described.
Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting.
It is understood that the examples and embodiments described herein are for illustrative purposes only and . . .
SUE~STITUTE SHEET
.... . . ... . . . .
WO91/09974 2 ~ PCT/US90/07313 that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
SlJBSTITUTE SHE~T
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~WO 91/0997~ 2 0 ~ 0 9 9 ~ P~/US90/07313 Z O H H H H H H Z H H
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E~ ~ ~ ~ O ~ ~ ~ ~ ~ ~ ~ O ~ ~
U~ IY H Cl 0 1--1 a ~ H ~ H ~ O H Cl 0 3 ~
~ W O 91/09974 . - PC~r/US90/07313 .
DIAGNOSTIC PROBE FOR DETECTING HUMAN STOMACH CANCER
A number of oncogene and proto-oncogene probes have been reported to be useful in diagnostic procedures for detecting certain forms of cancer and for following disease prognosis. However, a specific probe for the diagnosis of human stomach cancer has not heretofore been known or described.
It is, therefore, an object of the present invention to provide specific probes for detecting human stomach cancer. Such a unique probe came into being by the unexpected finding that the met proto-oncogene was activated in certain human cancer cell lines tested. The characteristics of the met gene can be found described in Park et al, 1986, Cell, 45:895-904; Park et al, 1987, PNAS, 84:6379-6383 and Gonzatti-Haces et al, 1988, PNAS, 85:21-25.
- Materials & Methods Various methodologies used for testing the activation of met gene are now described.
Southern Blottinq Genomic DNA from the HOS line and several gastric carcinoma cell lines were digested with EcoRI, ~5 fractionated on a 1% agarose gel, and transferred to nitrocellulose. The blot was baked for 2 hours. The blot was prehybridized for 4 hours at 42-C. The prehybridization and hybridization was in 50~
formamide, 5x Denhardts, 200 ~g/ml salmon sperm DNA, 5x SSPE, and 0.1% SDS. The blot was washed with 2x SSC, 0.1% SD5 for 20 minutes at RT, then with 3 washes of 0.2 x SSC, 0.1% SDS at 68-C.
.
.i - SUBSTITUTE SHEET
.,. . ~ .............. . . .
.
. . .
. ~ .
W O 91/09974 2 ~ ~ O ~ ~ ~ P~r/US90/07313 ~
Northern Blottina Total RNA was fractionated on a 1.1% formaldehyde-agarose gel and transferred to nitrocellulose. The nitrocellulose was baked for 2 hours, then washed 20 minutes in 5x SCC. The blot was prehybridized for 4 hours at 42C. Prehybridization and hybridization w~re in 50% formamide, 0.1% SDS, 6x SSPE, lx Denhardts, and 200 ~g/ml sonicated salmon sperm DNA. The blot was washed 2 times for 10 minutes in 2x SCC, 0.1% SDS at room temperature (about 22~-24C), then 3 times for 20 minutes each in 0.2x SSC, 0.1% SDS at 55C.
35S Metabolic Labeling and Immunoprecipitation Cells were preincubated 15 minutes in DMEM lacking methionine and cysteine supplemented with 10% calf serum, then labeled for 30 minutes in the above media supplemented with 2.5 mCi/ml 35S-methionine and cysteine. Cells were lysed and met was then immunoprecipitated with a mon~clonal directed against the kinase domain. Immunoprecipitates were fractionated on a 3-17% gradient gel. Proteins were detected by fluorography.
32p Metabolic Labelinq Cells were preincubated for 15 minutes with DMEM
lacking phosphate, then labeled for 2 hours in the above media supplemented with 0.5 mCi/ml 32p_ orthophosphate.
Monoclonal Antibody to the met Oncoaene Product A deposit of the hybridoma producing monoclonal antibody having specificity to the met oncogene product has been made at the ATCC, Rockville, MD., on December 14, 1989 under accession number HB 10309. The deposit shall be viably maintained, replacing if it becomes ,~ .
.. :
8VBSTITUT~ SHE~ F-r - . . . . .... . .- .. - . ::
. .
20~9~
~WO91/09974 PCT/US90/07313 non-viable during the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and upon issuance of the patent made available to the public without restriction in accordance with the provision of the law. The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
RESULTS
cFigure 1 shows the gene copy number in gastric carcinoma cell lines as determined by Southern blot. A
fragment representing most of the met cDNA was used to probe genomic DNA which had been digested with EcoRI.
Several hybridizing bands are detected. The MKN-45 and Okajima cell lines show siignificant amplification of the met gene. No rearrangement is detected with this probe. To further confirm these results, RNA levels were examined by Northern blot. Total RNA from several cell lines was probed with a fragment containing the met extracellular domain. A 9 kb met RNA is detected in all cell lines. As shown in Fig. 2, the met RNA is ~
greatly overexpressed in the MXN-45 and Okajima cell lines.
:......................................................................... , Then the expression and half-life of the met protein in some of these lines was examined by pulse chase. Cells were labeled with 35S for thirty minutes and then chased with cold media for l/2, 2 or 4 hours.
~/As shown in Fig. 3, very high levels of the met protein are detected in the MXN-45 line. The protein appears to be processed normally. The MXN-74 and KATOIII lines shown normal protein levels. The Okajima line shows the greatest amount of protein after a 30 minute ~, .
-, 1 SlJBSTlT~lTE SHEE r i - . ~ ..
- , ,- .
, . - . .. .. . -- - :, : . .
. . - . : ~ . - :
WO9~/09974 2 ~ ~ O 9 9 ~ PCT/US90/07313 ~
labeling, indicating a high rate of synthesis. While the met protein in this line is processed to the 140 form, the 140 is almost all gone after a four-hour chase, indicating a very short half-life.
It was then examined whether the proteins might be phosphorylated on tyrosine. Cells were labeled for 2 hours with 32p orthophosphate and protein was precipitated with an antibody directed against phosphotyrosine. As seen in Fig. 4, both the MKN-45 and Okajima lines show phosphotyrosine present on the pl40 form. No phosphotyrosine was detected on the pl40 in the other cell lines, even with a long overexposure.
Table l summarizes the data presented in Figs. 1-4.
Of the 7 lines examined, two (MKN-45 and Okajima) showed ampIification and phosphorylation on tyrosine on pl40. The Okajima line was unique in displaying a very ~;short half-life for pl40. When these lines were examined for their ability to grow in serum-free media, only the Okajima line was found competent to grow. It ~;20 is significant to note that met was found amplified only in poorly differentiated adenocarcinomas. The other lines examined for which a classification is known included two well differentiated adenocarcinomas ;and signet ring carcinomas.
The results indicate that the met gene is amplified and overexpressed only in poorly differentiated gastric carGinoma cell lines tested. The rapid turnover seen in one line may indicate that an autocrine loop is involved in the genesis of the tumor. Since the met amplification was seen only in poorly differentiated adenocarcinomas, clearly a met probe may be of great value in characterizing the clinical stage to which a ( :
SU E~STITUTE SH ~E~ .
:
-. . . . , . - . ,` - . ~ ~ :.- - .:
. .- - :
- . ` : . , :
~ 091/09974 2 ~ 7 0 9 9 ~ PCT/US90/07313 tumor has progressed. The availability of the monoclonal antibodies to the met gene product now makes ~-it possible not only to isolate and purify, but also to detect the presence of the met gene products in a biological sample by standard i~munological techniques including in situ immunofluorescence or other standard techniques. Accordingly, a diagnostic kit for the detection of met gene product, comprises a container containing antibodies to the met gene product.
lOOf course, given the nucleotide and amino acid sequences, a nucleic acid or polypeptide probe for detecting the met gene or the met gene product is easily made by conventional methodologies well known to one of ordinary skill in the art. Nucleic acid probes useful for this purpose re described in Park et al, supra, while probes are also described in Gonzatti-Haces et al, supra.
It is noted that unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
;Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials have been described.
Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting.
It is understood that the examples and embodiments described herein are for illustrative purposes only and . . .
SUE~STITUTE SHEET
.... . . ... . . . .
WO91/09974 2 ~ PCT/US90/07313 that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
SlJBSTITUTE SHE~T
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---
Claims (6)
1. A method for detecting clinical stages of human stomach cancer, comprising the step of determining the over expression of met gene in a human stomach tumor specimen by a suitable probe, an overexpression of met gene in said specimen being indicative of the cancerous stage.
2. A DNA fragment which binds specifically with the met gene.
3. An antibody which binds specifically with the met gene product.
4. A diagnostic kit for detecting the presence of met gene product in a biological sample, comprising a container containing the antibody of claim 3.
5. A method for detecting the presence of met gene product in a biological sample, comprising reacting a biological sample in which the presence of met gene product is to be determined, with the antibody of claim 3, a positive immunological reaction being indicative of the presence of met gene product in said sample.
6. A method for isolating purified met gene product, comprising the step of adsorbing met gene product utilizing the antibody of claim 3 and then recovering the adsorbed met gene product therefrom in a purified form.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US45755689A | 1989-12-27 | 1989-12-27 | |
| US457,556 | 1989-12-27 | ||
| PCT/US1990/007313 WO1991009974A1 (en) | 1989-12-27 | 1990-12-19 | Diagnostic probe for detecting human stomach cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2070995A1 true CA2070995A1 (en) | 1991-06-28 |
Family
ID=23817186
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002070995A Abandoned CA2070995A1 (en) | 1989-12-27 | 1990-12-19 | Diagnostic probe for detecting human stomach cancer |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0507868A4 (en) |
| JP (1) | JPH05504476A (en) |
| AU (1) | AU639532B2 (en) |
| CA (1) | CA2070995A1 (en) |
| WO (1) | WO1991009974A1 (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6673559B1 (en) | 1989-12-27 | 2004-01-06 | The Government Of The United States Of America As Represented By The Department Of Health And Human Services | Met proto-oncogene and a method for predicting breast cancer progression |
| ATE155525T1 (en) * | 1991-05-10 | 1997-08-15 | Pharmacia & Upjohn Spa | SHORTENED FORMS OF THE HEPATOCYTE GROWTH FACTOR (HGF) RECEPTOR |
| AU4645293A (en) * | 1992-06-26 | 1994-01-24 | United States Of America, Represented By The Secretary, Department Of Health And Human Services, The | The met proto-oncogene and a method for predicting breast cancer progression |
| DE4225569C2 (en) * | 1992-08-03 | 1996-04-25 | Max Planck Gesellschaft | Use of a probe for tumor diagnosis or tumor therapy |
| US20020068307A1 (en) * | 2000-03-30 | 2002-06-06 | Jason Pluta | Compositions and methods for diagnosing, monitoring, staging, imaging and treating stomach cancer |
| WO2002064839A2 (en) * | 2001-02-14 | 2002-08-22 | Tularik Inc. | Methods for the diagnosis and treatment of tumors employing the hepsin gene |
| DE10254601A1 (en) | 2002-11-22 | 2004-06-03 | Ganymed Pharmaceuticals Ag | Gene products differentially expressed in tumors and their use |
| DE102004024617A1 (en) | 2004-05-18 | 2005-12-29 | Ganymed Pharmaceuticals Ag | Differentially expressed in tumors gene products and their use |
| EP1790664A1 (en) | 2005-11-24 | 2007-05-30 | Ganymed Pharmaceuticals AG | Monoclonal antibodies against claudin-18 for treatment of cancer |
| EP1997832A1 (en) | 2007-05-29 | 2008-12-03 | Ganymed Pharmaceuticals AG | Monoclonal antibodies against Claudin-18 for treatment of cancer |
| PT3904391T (en) | 2010-03-10 | 2024-10-14 | Genmab As | Monoclonal antibodies against c-met |
| NZ707831A (en) | 2012-11-13 | 2018-11-30 | Astellas Pharma Inc | Agents for treatment of claudin expressing cancer diseases |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5015568A (en) * | 1986-07-09 | 1991-05-14 | The Wistar Institute | Diagnostic methods for detecting lymphomas in humans |
-
1990
- 1990-12-19 AU AU71406/91A patent/AU639532B2/en not_active Ceased
- 1990-12-19 WO PCT/US1990/007313 patent/WO1991009974A1/en not_active Application Discontinuation
- 1990-12-19 JP JP3503249A patent/JPH05504476A/en active Pending
- 1990-12-19 EP EP19910902835 patent/EP0507868A4/en not_active Withdrawn
- 1990-12-19 CA CA002070995A patent/CA2070995A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU639532B2 (en) | 1993-07-29 |
| WO1991009974A1 (en) | 1991-07-11 |
| AU7140691A (en) | 1991-07-24 |
| EP0507868A4 (en) | 1992-11-04 |
| JPH05504476A (en) | 1993-07-15 |
| EP0507868A1 (en) | 1992-10-14 |
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