Xiao Wei, Pacyna-Gengelbach Manuela, Schlüns Karsten, An Qian, Gao Yanning, Cheng Shujun, Petersen Iver
Institute of Pathology, Charité Medical School, Schumannstr. 20-21, 10098 Berlin, Germany.
Oncol Rep. 2005 Jul;14(1):229-34.
To investigate the gene expression of 9 cDNA clones generated by suppression subtraction hybridization (SSH), Northern blot analysis was performed on a panel of immortalized bronchial epithelial cell lines, lung cancer cell lines and normal human bronchial epithelial cells (HBEC). The clones were located on chromosomes 1q, 2q, 3p, 3q, 4q and 14q representing regions that are frequently affected by DNA imbalances as shown by comparative genomic hybridization (CGH). Two were unknown (H24, H103) whereas the others matched to the Pest-containing nuclear protein (H52), Rp11-767C1 gene (H134), the hypothetical gene AK025444 (H238), Guanine nucleotide binding protein (G protein)/alpha inhibiting activity polypeptide 2 (H268), Laminin gamma 1 (Y45), the DEAD (Asp-Glu-Ala-Asp/His) box polypeptide 9 (Y162) and the heat shock 90 kDa protein 1, alpha (Y238). Northern blot results indicated that all of the studied clones showed differential up- or down-regulation in immortalized cells and lung cancer cell lines. Of those, clone Y238 representing HSP90alpha showed a clearly over-expressed transcript. Subsequently, semi-quantitative RT-PCR was used to further confirm the over-expression of Y238, indicating that HSP90alpha was significantly over-represented in 49 primary lung tumors as compared to 14 normal lung samples (P<0.01). CGH showed that the majority of studied lung cancer cell lines (71.4%) carried an overrepresentation at 14q32 where HSP90alpha is located suggesting that it may be affected by DNA copy number changes. The further characterization of these clones will provide us with valuable information on its role in lung carcinogenesis and may help to develop new diagnostic or therapeutic targets for this lethal disease.
为了研究通过抑制性消减杂交（SSH）产生的9个cDNA克隆的基因表达，对一组永生化支气管上皮细胞系、肺癌细胞系和正常人支气管上皮细胞（HBEC）进行了Northern印迹分析。这些克隆位于1号染色体长臂、2号染色体长臂、3号染色体短臂、3号染色体长臂、4号染色体长臂和14号染色体长臂上，代表了比较基因组杂交（CGH）显示的经常受DNA失衡影响的区域。其中两个是未知的（H24、H103），而其他的分别与含Pest的核蛋白（H52）、Rp11 - 767C1基因（H134）、假定基因AK025444（H238）、鸟嘌呤核苷酸结合蛋白（G蛋白）/α抑制活性多肽2（H268）、层粘连蛋白γ1（Y45）、DEAD（天冬氨酸 - 谷氨酸 - 丙氨酸 - 天冬氨酸/组氨酸）框多肽9（Y162）和热休克90 kDa蛋白1α（Y238）匹配。Northern印迹结果表明，所有研究的克隆在永生化细胞和肺癌细胞系中均显示出差异上调或下调。其中，代表HSP90α的克隆Y238显示出明显过表达的转录本。随后，使用半定量RT - PCR进一步证实Y238的过表达，表明与14个正常肺样本相比，HSP90α在49个原发性肺肿瘤中显著过量表达（P<0.01）。CGH显示，大多数研究的肺癌细胞系（71.4%）在HSP90α所在的14q32处存在过量表达，提示其可能受DNA拷贝数变化影响。对这些克隆的进一步表征将为我们提供有关其在肺癌发生中作用的有价值信息，并可能有助于为这种致命疾病开发新的诊断或治疗靶点。
