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Diagnostic method for cancer characterized in the detection of the deletion of G-CSF exon 3

a cancer and exon technology, applied in the field of cancer diagnosis, can solve the problems of generating false positive data, morphological and immunohistochemical diagnosis requires much longer time, and high cost, and achieves the effect of simple and rapid diagnosis

Inactive Publication Date: 2005-07-21
LEE SANG +6
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006] Leading to the present invention, the thorough and intensive research into a cancer biomaker capable of diagnosing a variety of cancers, conducted by the present inventors, resulted in the finding that exon 3 skipping occurs during transcription of the G-CSF gene in cancer patients, and use of G-CSF mRNA fragment or protein as a diagnostic cancer marker can achieve diagnosis of a variety of cancer, wherein the diagnosis is performed simply and quickly, as well as being economical.

Problems solved by technology

Cancer is a leading cause of death in developed nations.
In comparison with the molecular diagnosis, the morphological and immunohistochemical diagnosis requires much longer time and higher cost.
However, the protein chip system does not use only a biomarker to diagnose all kinds of cancer, but uses 10 or more proteins.
The known cancer biomarkers which have low organ specificity, such as CEA, BFP, TPA and IAP, also, have low sensitivity, thus generating false positive data.

Method used

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  • Diagnostic method for cancer characterized in the detection of the deletion of G-CSF exon 3
  • Diagnostic method for cancer characterized in the detection of the deletion of G-CSF exon 3
  • Diagnostic method for cancer characterized in the detection of the deletion of G-CSF exon 3

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of mRNA and cDNA from Tumor Cell Lines

[0055] mRNA and cDNA samples were prepared from 8 normal cell lines and tissues, and 17 tumor cell lines. The normal cell lines and tumor cell lines used in Examples of the present invention are given in Table 1, below.

Table 1

Normal and Tumor Cell Lines Used in the Present Invention

[0056]

Cell typesCell collection centersTumor cellYCC-7Stomach cancer cell lineCancer metastasis research center,linesCollege of Medicine, Yonsei UniversityAGSStomach cancer cell lineATCC CRL-1739SNU-1Stomach cancer cell lineKorean Cell Line Research Foundation(KCLRF), Seoul National UniversityMDA-MB-Breast cancer cell lineATCC HTB-26231MCF-7Breast cancer cell lineATCC HTB-22SK-BR-3Breast cancer cell lineATCC HTB-30HT-1080Sarcoma cell lineATCC CCL-121HCT-116Colon cancer cell lineATCC CCL-247COLO205Colon cancer cell lineATCC CCL-222DLD-1Colon cancer cell lineATCC CCL-221HT-29Colon cancer cell lineATCC HTB-38A549Lung cancer cell lineATCC CCL-185NCI-H460Lu...

example 2

Detection of hG-CSF Gene by PCR

[0060] In order to detect expression of normal hG-CSF gene in each tumor cell line, PCR was carried out using cDNA prepared in Example 1 as a template. As shown in FIG. 2, PCR reactions were divided into three types according to their amplified products, as follows: PCR 1 for amplification of a region (Thr1-Pro174) ranging from a part of exon 2 to exon 5 of hG-CSF gene; PCR 2 for amplification of a region (Ile24-Leu71) ranging from a part of exon 2 to exon 3 of hG-CSF gene; and PCR 3 for amplification of a region (Cys36-Ser80) ranging from exon 3 to a part of exon 4 of hG-CSF gene.

[0061] PCR 1 was carried out using a cDNA sample from each tumor cell line as a template, and a primer set of a sense primer designated SEQ ID NO.: 1 (5′-ACCCCCCTGGGCCCTGCC-3′) and an antisense primer designated SEQ ID NO.: 2 (5′-TCAGGGCTGGGCAAGGTG-3′). PCR 2 was carried out using a cDNA sample from each tumor cell line as a template, and a primer set of a sense primer desi...

example 3

Detection of G-CSF Gene by Hybridization

[0065] The deletion of exon 3 in G-CSF cDNA from tumor cell lines was detected by hybridization.

[0066] PCR was carried out using G-CSF gene derived from a normal cell line as a template, and a primer set of a sense primer designated SEQ ID NO.: 4 (5′-TGTGCCACCTACAAGCTGTGC-3′) and an antisense primer designated SEQ ID NO.: 5 (5′-CAGCTGCAGGGCCTGGCT-3′). A DNA fragment of 108 bp corresponding to the exon 3 region of G-CSF gene was obtained.

[0067] Separately, PCR was carried out using G-CSF gene derived from the normal cell line as a template, and a primer set of a sense primer designated SEQ ID NO.: 1 (5′-ACCCCCCTGGGCCCTGCC-3′) and an antisense primer designated SEQ ID NO.: 9 (5′-CAGCTTCTCCTGGAGCGC-3′). A DNA fragment of 105 bp corresponding to the exon 2 region of G-CSF gene was obtained.

[0068] After being purified, each of the DNA fragments (50 ng / μl) was spotted on a nylon membrane (Boehringer Mannheim, Germany), and incubated at 80° C. fo...

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Abstract

Disclosed are a method, a composition, a microarray, an antibody and a kit for diagnosis and prognosis of cancer, based on detection of deletion of the exon 3 region of G-CSF gene or levels of a mutated G-CSF protein having a deletion of an amino acid sequence corresponding to the exon 3 region, wherein the deletion of the exon 3 region of the G-CSF gene is used as a cancer biomarker.

Description

CONTINUING DATA [0001] The present application is a divisional application of 10 / 490,502, filed Mar. 22, 2004.TECHNICAL FIELD [0002] The present invention relates to a method of diagnosing cancer based on modified features in granulocyte colony stimulating factor (G-CSF) mRNA or protein. More particularly, the present invention relates to a diagnostic and prognostic method for cancer based on skipping of the exon 3 region of the G-CSF gene at mRNA or protein levels, wherein skipping of G-CSF exon 3 is used as a diagnostic cancer marker. PRIOR ART [0003] Cancer is a leading cause of death in developed nations. For this reason, a major interest in cancer therapy is to develop methods for early diagnosis and treatment of cancer. Typically, late-stage cancer is almost incurable, whereas, at the early stage, cancer can be more effectively treated and therapeutic methods for early-stage cancer are simpler. Therefore, there is an urgent need for development of methods for accurately and qu...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/535C07K16/00C07K16/24C12M1/00G01N33/53C12N15/02C12N15/09C12N15/12C12P21/08C12Q1/68G01N33/566G01N37/00
CPCC07K16/243C12Q1/6886C12Q2600/156C12N15/11
Inventor LEE, SANGYOO, NAEYOO, SOCHUNG, HYUNKEUM, KIYOO, WONJEONG, KI
Owner LEE SANG
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