WO1991009974A1

WO1991009974A1 - Diagnostic probe for detecting human stomach cancer

Info

Publication number: WO1991009974A1
Application number: PCT/US1990/007313
Authority: WO
Inventors: Thomas Edward Kmiecik; George Franklin Vande Woude
Original assignee: The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce
Priority date: 1989-12-27
Filing date: 1990-12-19
Publication date: 1991-07-11
Also published as: EP0507868A4; CA2070995A1; AU639532B2; EP0507868A1; AU7140691A; JPH05504476A

Abstract

A diagnostic probe for detecting human stomach cancer is described.

Description

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_f 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 Blotting

Geno ic DNA from the HOS line and several gastric carcinoma cell lines were digested with EcoRI, 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 μg/ml salmon sperm DNA, 5x SSPE, and 0.1% SDS. The blot was washed with 2x SSC, 0.1% SDS for 20 minutes at RT, then with 3 washes of 0.2 X SSC, 0.1% SDS at 68°C.

SUBSTITUTE SHEET Northern Blotting

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 42°C. Prehybridization and hybridization were in 50% formamide, 0.1% SDS, 6x SSPE, lx Denhardts, and 200 μg/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°-24^,'C), then 3 times for 20 minutes each in 0.2x SSC, 0.1% SDS at 55"C. 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 monoclonal directed against the kinase domain. Immunoprecipitates were fractionated on a 3-17% gradient gel. Proteins were detected by fluorography. ³²P Metabolic Labeling

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 Oncogene 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

SUBSTITUTE SHEET 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 Figure 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 significant 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 MKN-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 1/2, 2 or 4 hours. As shown in Fig. 3, very high levels of the met protein are detected in the MKN-45 line. The protein appears to be processed normally. The MKN-74 and KATOIII lines shown normal protein levels. The Okajima line shows the greatest amount of protein after a 30 minute

SUBSTITUTESHEET 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 1 summarizes the data presented in Figs. 1-4. Of the 7 lines examined, two (MKN-45 and Okajima) showed amplification 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 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 carcinoma 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

SUBSTITUTE SHEET 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 immunological 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. Of 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 are described in Park et al, supra_f 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

SUBSTITUTE SHEET 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.

SUBSTITUTE SHEET TABLE 1

CEll MET pl40 SHORT pl40 SERUM FREE LINE AMPLIFIED HALF LIFE P-TYR GROWTH CLASSIFICATION

HOS OSTEOSARCOMA

Claims

WHAT IS CLAIMED IS:

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.

SUBSTITUTE SHEET