ETIOLOGY OF KARYOTYPIC DEFECTS IN ORAL CANCER

口腔鳞状癌（OSCC）的特征是复杂的，通常是近三倍体核型，具有多个数字和结构异常。据报道，口腔癌的非整倍体发生率高于子宫癌、宫颈癌、结直肠癌或乳腺癌。在大多数OSCC细胞系中，细胞在克隆染色体改变的背景下表现出结构和数值变化。因此，染色体不稳定性（CIN）可能在OSCC中起关键作用，使其成为一种优秀的检测系统。口腔癌中CIN高发生率和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的一个重要因素。我们的初步结果支持我们的假设。OSCC细胞的检查显示多极纺锤体，染色体滞后，纺锤体结构异常，微核频率高。为了研究CIN在OSCC中的作用机制，我们将把我们的OSCC细胞系与正常的人口腔角质细胞（NHOK）培养物进行比较，以满足以下特定目的：1)通过免疫细胞化学与关键细胞骨架蛋白抗体和经典分子细胞遗传学分析，依次表征同一OSCC细胞中与对照NHOK相比的细胞遗传学和细胞骨架变化；2)利用印迹法和测序法，根据选定的细胞骨架蛋白的表达改变和/或基因畸变，确定OSCC细胞骨架缺陷的基础；(3)通过改变NHOKs中细胞骨架蛋白的水平，并评估这种变化是否导致CIN，确定所观察到的细胞骨架蛋白的变化是否与CIN有因果关系。本研究预计将首次提供明确的证据，证明细胞骨架功能障碍是OSCC中观察到的CIN的主要因素，并将阐明OSCC细胞中分离改变的方式，并将识别特异性细胞骨架蛋白，其表达改变可能导致OSCC中CIN的发生。这些蛋白中的一种或多种表达的改变也可能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可以纠正分离缺陷，它可能被证明是减少肿瘤中CIN的有效手段，因此，具有大量，多样，不可修复的遗传改变的异质性可能是徒劳的。鉴定携带CIN的细胞之后，将能够应用引导细胞自然死亡的治疗方法。

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.