ETIOLOGY OF KARYOTYPIC DEFECTS IN ORAL CANCER

口腔鳞状细胞癌（OSCC）的特点是复杂的，往往近三倍体核型与多个数量和结构异常。据报道，口腔癌中的非整倍体频率高于子宫癌、宫颈癌、结肠直肠癌或乳腺癌。在大多数OSCC细胞系中，细胞在克隆染色体改变的背景下显示结构和数量变化。因此，染色体不稳定性（CIN）可能在OSCC中发挥关键作用，使其成为一个优秀的测试系统。口腔癌中CIN的高发生率和由此导致的非整倍体的原因是什么？我们假设关键纺锤体、中心体和着丝粒（细胞骨架）蛋白的改变是OSCC中CIN的重要促成因素。我们的初步结果支持我们的假设。口腔鳞状细胞癌细胞的检查显示多极纺锤体，落后的染色体，异常的纺锤体结构，和高频率的微核。为了研究CIN在OSCC中的机制，我们将比较我们的OSCC细胞系与正常人口腔角质形成细胞（NHOK）培养物以满足以下特定目的：1）通过免疫细胞化学用针对关键细胞骨架蛋白的抗体和经典分子细胞遗传学分析，依次表征与对照NHOK相比相同OSCC细胞中的细胞遗传学和细胞骨架改变，2）使用印迹和测序，根据所选细胞骨架蛋白的表达改变和/或其基因中的畸变，确定OSCC中细胞骨架缺陷的基础，以及（3）通过改变NHOK中细胞骨架蛋白的水平，并评估是否观察到细胞骨架蛋白的变化与CIN有因果关系，这种变化导致CIN。 这项研究预计将产生第一个明确的证据表明，细胞骨架功能障碍是一个主要的贡献者在口腔鳞状细胞癌中观察到的CIN，将澄清的方式，其中分离是改变在口腔鳞状细胞癌细胞，并将确定特定的细胞骨架蛋白，改变表达可能有助于在口腔鳞状细胞癌的CIN。这些蛋白质中的一种或多种的改变的表达也可以作为口腔粘膜或肿瘤细胞中CIN的有用生物标志物，可能用于早期检测。如果重新建立正常的有丝分裂蛋白水平纠正分离缺陷，它可能被证明是一种有效的手段，尽量减少肿瘤中的CIN，因此，异质性与众多，不同的，不可挽回的遗传变异，可能是徒劳的。然后，携带CIN的细胞的鉴定将使得能够应用引导细胞进入自然细胞死亡的疗法。

Oral squamous carcinomas (OSCC) are characterized by complex, often near-triploid karyotypes with multiple numerical and structural abnormalities. The frequency of aneuploidy in oral cancers is reportedly higher than uterine, cervical, colorectal or breast cancers. In most OSCC cell lines, cells display structural and numerical variations on a background of clonal chromosomal alterations. Thus, chromosomal instability (CIN) may play a key role in OSCC, making it an excellent test system. What is the cause of this high rate of CIN and resulting aneuploidy in oral cancer? We hypothesize that alterations in key spindle, centrosomal, and centromeric (cytoskeletal) proteins are an important contributing factor to the CIN in OSCC. Our preliminary results support our hypothesis. Examination of OSCC cells reveals multipolar spindles, lagging chromosomes, abnormal spindle structure, and a high frequency of micronuclei. To investigate the mechanism of CIN in OSCC, we will compare our OSCC cell lines to normal human oral keratinocyte (NHOK) cultures to satisfy the following Specific Aims, to 1) sequentially characterize the cytogenetic and cytoskeletal alterations in the same OSCC cells compared to control NHOKs by immunocytochemistry with antibodies to key cytoskeletal proteins and classical molecular cytogenetic analyses, 2) define a basis for the cytoskeletal defects in OSCC in terms of altered expression of selected cytoskeletal proteins and/or aberrations in their genes using blotting and sequencing, and (3) determine whether the observed changes in cytoskeletal proteins are causally related to CIN by altering the levels of the cytoskeletal proteins in NHOKs and assessing whether this change causes CIN. This study is expected to produce the first clear-cut evidence that cytoskeletal dysfunction is a major contributor to the observed CIN in OSCC, will clarify the manner in which segregation is altered in OSCC cells, and will identify specific cytoskeletal proteins, altered expression of which may contribute to the CIN in OSCC. Altered expression of one or more of these proteins may also serve as a useful biomarker of CIN in oral mucosal or tumor cells, possibly for early detection. If re-establishing normal mitotic protein levels corrects segregation defects, it may prove to be an effective means of minimizing CIN in tumors, thus, heterogeneity with numerous, varied, irreparable genetic alterations, may be fruitless. Identification of cells bearing CIN will then enable application of therapies that guide the cells into a natural cell death.