Hoa Michael, Davis Shannon L, Ames Sarah J, Spanjaard Remco A
Department of Otolaryngology-Head and Neck Surgery, Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Cancer Res. 2002 Dec 15;62(24):7154-6.
In contrast to many other tumors of different lineage, oncogenic ras mutations are rarely found within head and neck squamous cell carcinoma (HNSCC). On the other hand, increased expression of wild-type K-ras in HNSCC tumor material has been noticed, but the potential physiological consequences of this observation have not yet been experimentally assessed. The current study addresses this issue by modulating K-ras expression in HNSCC cell lines and primary keratinocytes and determining its effects on cell growth and survival in vitro. Consistent with earlier reports using patient tumor material, Western blot analysis of four HNSCC lines (SCC-9, SCC-15, SCC-25, and FaDu) revealed varying but universally increased protein expression of K-ras relative to keratinocytes. All HNSCC lines expressed wild-type K-ras mRNA based on a random sequencing of eight K-ras cDNA samples obtained by reverse transcription-PCR from each HNSCC line (P <or= 0.00391). Transfection of keratinocytes with a plasmid expression vector containing wild-type K-ras cDNA resulted in dramatically increased proliferation and survival compared with control-transfected or untransfected keratinocytes. Conversely, transfection of FaDu cells, which express the highest level of endogenous K-ras, with K-ras antisense oligonucleotides but not control oligonucleotides significantly reduced cellular proliferation (P <or= 0.0022). These results show that the level of K-ras protein expression is a major determinant of proliferation of HNSCC cells and keratinocytes and suggest that amplification of nonmutated K-ras in HNSCC contributes to tumor growth. These novel findings may have important ramifications for potential K-ras-targeted interventions in the treatment of HNSCC.
与许多不同谱系的其他肿瘤相比，致癌性ras突变在头颈部鳞状细胞癌（HNSCC）中很少见。另一方面，已注意到HNSCC肿瘤组织中野生型K-ras的表达增加，但这一观察结果的潜在生理后果尚未经过实验评估。本研究通过调节HNSCC细胞系和原代角质形成细胞中K-ras的表达，并确定其对体外细胞生长和存活的影响来解决这一问题。与早期使用患者肿瘤组织的报告一致，对四个HNSCC细胞系（SCC-9、SCC-15、SCC-25和FaDu）进行的蛋白质印迹分析显示，相对于角质形成细胞，K-ras的蛋白质表达存在差异但普遍增加。基于从每个HNSCC细胞系通过逆转录-PCR获得的八个K-ras cDNA样本的随机测序，所有HNSCC细胞系均表达野生型K-ras mRNA（P≤0.00391）。用含有野生型K-ras cDNA的质粒表达载体转染角质形成细胞，与对照转染或未转染的角质形成细胞相比，增殖和存活率显著增加。相反，用K-ras反义寡核苷酸而非对照寡核苷酸转染内源性K-ras表达水平最高的FaDu细胞，可显著降低细胞增殖（P≤0.0022）。这些结果表明，K-ras蛋白表达水平是HNSCC细胞和角质形成细胞增殖的主要决定因素，并提示HNSCC中非突变K-ras的扩增有助于肿瘤生长。这些新发现可能对HNSCC治疗中潜在的K-ras靶向干预具有重要意义。
