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Genomic analysis in oral squamous cell carcinoma detected by restriction landmark genomic scanning

通过限制性标志基因组扫描检测口腔鳞状细胞癌的基因组分析

基本信息

批准号：
10671899
负责人：
SUGIMURA Masahito
金额：
$ 1.6万
依托单位：
Nara Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671899/
关键词：
Oral Squamous cell carcinoma Genome RLGS DNA

项目摘要

Although a considerable amount of cytogenetic and molecular data on oral squamous cell carcinoma (OSCC) has accumulated over the last few years, the picture is still incomplete in terms of how genetic alterations are involved in tumor initiations and progression. We, therefore, examined genetic abnormalities in OSCC using restriction landmark genomic scanning (RLGS), a method of two-dimensional gel analysis allowing detection of amplifications and other aberrations in genomic DNA. At first, DNA extracted from each of four oral carcinoma cell lines (Ca9-22, HO-1-u-1, HSC-2, KB) was examined. Isolated cell line and normal oral epithial DNAs were sequentially cleaved with specific restriction enzymes, radiolabelled and separated in two-dimensional gel electrophoresis. Thirteen distinct fragments were commonly amplified in the oral cancer cell lines, six of which were evident in all samples. Secondly, eleven cases of OSCC were also examined in the same manner. Five fragment/spots were found and amplified in at least 64% (7/11) (chromosomenos.4,9-12 or 22) of carcinomas, with one of these sports amplified in 100% (chromosome no.4) of tumor samples. In addition, six other spots were frequently reduced in at least 55% (chromosome nos.15 or 9-12) of tumor tissues. Then, the question of whether the DNA alterations involve not only genetic but also epigenetic change was examined using cell lines-Ca9-22, HO-1-u1, HSC-2 and KB-and treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-AzaCDR). Intensities of two of the reduced sports were amplified in the cell lines by the 5-AzaCDR treatment, showing that they were due to altered DNA methylation. The current studies provide clear evidence that epigenetic changes, like the methylation are related to oral carcinogenesis.

虽然在过去的几年里，口腔鳞状细胞癌（OSCC）的细胞遗传学和分子生物学数据已经积累了相当多的，图片仍然是不完整的遗传变异是如何参与肿瘤的发生和发展。因此，我们使用限制性标志基因组扫描（RLGS），一种二维凝胶分析方法，可以检测基因组DNA中的扩增和其他畸变，检查口腔鳞癌的遗传异常。首先，检测从4种口腔癌细胞系（Ca 9 -22、HO-1-u-1、HSC-2、KB）中提取的DNA。分离的细胞系和正常口腔上皮DNA依次用特异性限制性内切酶切割，放射性标记并在二维凝胶电泳中分离。口腔癌细胞系中通常扩增出13个不同的片段，其中6个片段在所有样本中都很明显。其次，还以同样的方式检查了11例OSCC。在至少64%（7/11）（4号、9 -12号或22号染色体）的癌中发现并扩增了5个片段/点，其中一个片段/点在100%（4号染色体）的肿瘤样品中扩增。此外，在至少55%的肿瘤组织（15号或9-12号染色体）中，其他6个斑点经常减少。然后，使用细胞系Ca 9 -22、HO-1-u1、HSC-2和KB并用去甲基化剂5-氮杂-2 '-脱氧胞苷（5-AzaCDR）处理，检查DNA改变是否不仅涉及遗传变化，而且涉及表观遗传变化。通过5-AzaCDR处理，在细胞系中扩增了两种减少的运动的强度，表明它们是由于改变的DNA甲基化。目前的研究提供了明确的证据表明，表观遗传变化，如甲基化与口腔癌的发生有关。