Rohatgi Nidhi, Kaur Jatinder, Srivastava Anurag, Ralhan Ranju
Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nager, New Delhi.
Oral Oncol. 2005 Sep;41(8):806-20. doi: 10.1016/j.oraloncology.2005.04.010.
Smokeless tobacco (ST) usage is a growing public health problem worldwide. Exposure to smokeless tobacco is carcinogenic to humans. The molecular mechanism(s) underlying ST associated oral carcinogenesis remain largely unknown. The major challenge is to identify the key factor(s) involved in malignant transformation of oral lesions. Knowledge of these factors will provide candidate diagnostic biomarkers and targets for early intervention. To identify the molecular targets in ST associated oral lesions, we established and purified cultures of epithelial cells (AMOL-III) from an oral leukoplakia with histological evidence of hyperplasia with hyperkeratosis from gingivo-buccal sulcus of a smokeless tobacco (khaini) consumer. Cell cultures were characterized and modulation of gene expression in response to smokeless tobacco extract (STE) was investigated using confocal microscopy and immunoblotting. AMOL-III cells showed altered expression of cell cycle regulators namely p53, p21waf1/cip1, hdm2, proliferation marker Ki67 and transcription factor Ets-1. These cells did not harbor HPV 16/18. No mutation was detected in H-Ras codon 12/13 or in p53 exons 5-9 in AMOL-III cells. STE treatment of these cells resulted in loss of pRb, RARbeta, p21 waf1/cip1 and O6-methyl guanine-DNA methyl transferase (MGMT) while the expression of cyclin D1 was increased. To our knowledge this is the first report to demonstrate that khaini modulates expression of multiple cellular targets including proteins involved in cell cycle regulation and DNA methylation, which may lead the oral epithelial cells down the carcinogenic pathway. This in vitro model system assumes importance in unraveling the cellular and molecular mechanisms implicated in smokeless tobacco associated early oral cancer progression.
无烟烟草（ST）的使用在全球范围内是一个日益严重的公共卫生问题。接触无烟烟草对人类具有致癌性。无烟烟草相关口腔癌发生的分子机制在很大程度上仍不清楚。主要挑战是确定参与口腔病变恶性转化的关键因素。了解这些因素将为早期干预提供候选诊断生物标志物和靶点。为了确定无烟烟草相关口腔病变中的分子靶点，我们从一名无烟烟草（嚼烟）使用者牙龈颊沟处的口腔白斑建立并纯化了上皮细胞培养物（AMOL-III），该白斑具有增生伴角化过度的组织学证据。对细胞培养物进行了表征，并使用共聚焦显微镜和免疫印迹研究了其对无烟烟草提取物（STE）的基因表达调节。AMOL-III细胞显示细胞周期调节因子p53、p21waf1/cip1、hdm2、增殖标志物Ki67和转录因子Ets-1的表达发生改变。这些细胞未携带HPV 16/18。在AMOL-III细胞的H-Ras密码子12/13或p53外显子5-9中未检测到突变。STE处理这些细胞导致pRb、RARβ、p21 waf1/cip1和O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）丢失，而细胞周期蛋白D1的表达增加。据我们所知，这是第一份证明嚼烟可调节包括参与细胞周期调节和DNA甲基化的蛋白质在内的多个细胞靶点表达的报告，这可能会使口腔上皮细胞走上致癌途径。这个体外模型系统对于阐明无烟烟草相关早期口腔癌进展所涉及的细胞和分子机制具有重要意义。
