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

尽管在过去的几年中，口腔鳞状细胞癌(OSCC)已经积累了大量的细胞遗传学和分子数据，但关于基因改变如何参与肿瘤的发生和发展，这一图景仍然不完整。因此，我们使用限制性地标基因组扫描(RLGS)检查了口腔鳞状细胞癌的遗传异常，这是一种二维凝胶分析方法，可以检测基因组DNA中的扩增和其他异常。首先，对四种口腔癌细胞系(Ca9-22、HO-1-u-1、HSC-2、KB)的DNA进行检测。分离的细胞系和正常口腔上皮DNA用特定的限制性内切酶顺序切割，放射性标记，双向凝胶电泳法分离。在口腔癌细胞系中普遍扩增出13个不同的片段，其中6个片段在所有样本中都明显存在。其次，对11例口腔鳞状细胞癌也进行了同样的检查。至少在%(7/11)(4、9-12或22号染色体)的癌组织中发现并扩增了5个片段/斑点，其中1个片段/斑点在100%(4号染色体)的肿瘤标本中扩增。此外，在至少55%的肿瘤组织(15号染色体或9-12号染色体)中，其他6个点经常发生减少。然后，使用细胞系Ca9-22、HO-1-U1、HSC-2和KB以及5-氮杂-2‘-脱氧胞苷(5-AzaCDR)处理来研究DNA改变是否不仅涉及基因改变，还包括表观遗传改变。经5-AzaCDR处理后，细胞系中两个减少的运动的强度被放大，表明它们是由于DNA甲基化的改变而引起的。目前的研究提供了明确的证据表明，表观遗传学变化，如甲基化与口腔癌的发生有关。

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.