| Project/Area Number |
63570147
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Human pathology
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| Research Institution | Shiga University of Medical Science (1989)
Kyoto Prefectural University of Medicine (1988) |
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Principal Investigator |
HAMADA Shinshichi Shiga University of Medical Science, Department of Pathology, Instructor, 医学部助手 (00172974)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1988: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Colorectal carcinoma / DNA aneuploidy / Gene amplification / Formalin-fixed and paraffin-embedded tissue / Ki-ras gene / ホルマリン固定パラフィン包埋組織 / Kiーras遺伝子 / ヒト大腸癌 / 細胞核DNA顕微蛍光測光法 / 異倍体癌 / Kーras遺伝子 / dot blot解析 / Southern blot解析 |
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| Research Abstract |
DNA aneuploidy is found in approximately 80% of advanced colorectal carcinomas. The purpose of this research is to detect DNA aneuploidy and gene amplification simultaneously in colorectal tumors with using formalin-fixed and paraffin-embedded pathology materials. This research project consists of following 3 steps. (1)Improvement for precise and reliable detection of DNA aneuploidy, (2)inerease in yield of isolated cells and DNA extracted from the materials and (3)comparative analysis of DNA ploidy and gene amplification. Relationships analyzed between fluorescence intensity, thickness of samples, DNA concentration and dye concentration revealed that the major factors resulting in measurement errors are uneven thickness of cell smears and difference in chromatin DNA concentration. It was required for measurement of DNA content with good linearity and a small coefficient of variation(CV) to make cell smears flattened homogeneously less than 0.3mum and to decrease dye concentration. The CV was decreased to 2-3% by these procedures even with formalin-fixed and paraffin-embedded materials.
Various enzymatic and mechanical treatments were studied to obtain isolated cells and DNA sufficiently from small materials. Sufficient cells were obtained constantly from formalin-fixed and paraffin-embedded tissues, even from minute carcinomas less than 1mm in diameter, with using proteinase K and tefron homogenizers. DNA can be also extracted from these materials.
DNA ploidy and amplification of ras family genes were studied in colorectal carcinomas and adenomas. Ki-ras-1 pseudogene was amplified in some cases of aneuploid careinomas(3 of 14 aneuploid cartinomas). All the adenomas were diploid and did not show its amplification. Ki-ras-2 was not amplified in both carcinomas and adenomas. No definite relationship was found between DNA aneuploidy and amplification of Ki-ras genes, although DNA aneuploidy was incidentally associated with its amplification during cancer progression.
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