松原 長秀 岡山大学, 医学部・附属病院, 医員
OGURA Hajime OKAYAMA UNIV.MEDICAL SCHOOL,Associate Professor, 医学部, 助教授 (20112146)
OUCHIDA Mamoru OKAYAMA UNIV.MEDICAL SCHOOL,Associate Professor, 医学部, 助教授 (80213635)
MATSUBARA Nagahide OKAYAMA UNIV.HOSPITAL,Medical Associate
|Budget Amount *help
¥7,600,000 (Direct Cost : ¥7,600,000)
Fiscal Year 1997 : ¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1996 : ¥5,300,000 (Direct Cost : ¥5,300,000)
As a prerequisite of this research, we established a tumor bank containing about 800 pairs of the specimens from a wide variety of human tumor patients during the term of this project. Using these materials, we have developed two novel approaches for detecting tumor-specific genetic alterations, one is the comprehensive genomic scanning method with inter-Alu long PCR amplification, and the other is the multiple SSCP analysis of the whole region of a gene.
On application of these novel techniques, we found a novel tumor-specific mutation in the gene encoding E2F4 transcription regulator in a subset of genetically unstable (MI+) colorectal cancers. The mutation was decrease in CAG repeat number within a coding exon of the gene, giving rise to reduction of one or two amino acids of 13 consecutive serine residues of the E2F4 protein. The incidence of this mutation in MI+ colorectal/gastric cancers was approximately 40% in both Japan and American patients. Next, we found that the mutation ap
pears to be a result of the inactivating frame-shift mutation of the hMSH3 gene, a DNA mismatch-repair gene implicated in the repair process of mismatch-loops of two to four nucleotides. The correlation between these two mutations was above 80 % (P = 0.0015 by Fisher's exact test). The E2F4 gene is known to behave as an active oncogene when it is overexpressed in living cells both in vivo and in vitro. Further, we got a preliminary result that the altered version of E2F4 gene is capable of stimulating cellular proliferation in rodent cell system. Thus, the E2F4 gene is identified, for the first time, as a target of the defective mismatch repair function involving hMSH3 protein, in actual human malignancies.
As a relevant research result, we have found novel genetic alterations of the human p107 gene, a close relative of the tumor suppressor gene RB.The most clear case of the genetic alteration of the gene, detected in a diffuse-large B cell lymphoma cell line, was an interstitial deletion of a 15 kbp region containing 5 coding exons of the gene. In contrast to the RB gene, tumor-specific alteration of the p107 gene has never been identified as far. Our finding in human hematologic malignancies is the first discovery of such cases. We analyzed the entire genomic structure of the p107 gene and found that the gene spans about 90 kbp region and is composed of total 22 coding exons.
Other results of our research projects include, 1) establishment of a general strategy for measuring gene expression by competitive PCR analysis, 2) identification of a novel activating mutation at 22nd codon of the K-ras gene in a human colon cancer, 3) discovery of a novel candidate of the tumor suppressor gene residing on chromosome 3p21, which may be related to many types of solid tumors. Less