Grendys E C, Barnes W A, Weitzel J, Sparkowski J, Schlegel R
Division of Gynecologic Oncology, Moffitt Cancer Center, University of South Florida, Tampa 33612, USA.
Gynecol Oncol. 1997 May;65(2):343-7. doi: 10.1006/gyno.1997.4649.
The ras oncogenes, Harvey (H), Kirsten (K), and neuroblastoma (N), are a family of genes coding for a membrane-associated protein (p21) which possesses inherent guanine triphosphatase (GTPase) activity. Point mutagenesis at codons 12, 13, and 61 has been implicated in ras activation and subsequent cellular transformation. Given the epidemiologic relationship of HPV infection with cervical carcinoma and the tumorigenic interaction of HPV and mutated ras oncogenes, this study was undertaken to identify if mutated ras oncogenes were present in early invasive cervical carcinomas.
A combination of polymerase chain reaction (PCR) and dot-blot hybridization was used to determine the frequency and types of ras point mutants occurring in cervical carcinoma. Thirty-three patients with early-stage cervical carcinoma were identified. DNA was extracted from archival tumor samples. ras genes were PCR amplified using flanking primers and hybridized with a series of labeled allele-specific oligonucleotides corresponding to wild-type forms of K12,61, N12,13,61, and H12,61, as well as to all combinations of substitution mutations (7 wild-type, 45 mutants).
ras mutations were identified in 24.2% of specimens. The detected mutations in H, K, and N-ras all occurred at codon 61. This was not the result of PCR or hybridization artifact in that mutations were detected in position 12 and 13 in appropriate control samples.
Mutant ras has been shown to convert HPV immortalized keratinocytes to the tumorigenic state. Our results indicate that a significant percentage (24.2%) of these early-stage cervical cancers contain activated ras. Additional studies will be needed to evaluate whether codon 61 represents a characteristic "hot-spot" of ras mutation in a subset of cervical carcinoma.
哈维（H）、 Kirsten（K）和神经母细胞瘤（N）的ras癌基因是一族编码膜相关蛋白（p21）的基因，该蛋白具有内在的鸟嘌呤三磷酸酶（GTPase）活性。密码子12、13和61处的点突变与ras激活及随后的细胞转化有关。鉴于人乳头瘤病毒（HPV）感染与宫颈癌的流行病学关系以及HPV与突变的ras癌基因的致瘤相互作用，本研究旨在确定早期浸润性宫颈癌中是否存在突变的ras癌基因。
采用聚合酶链反应（PCR）和点杂交相结合的方法来确定宫颈癌中ras点突变的频率和类型。确定了33例早期宫颈癌患者。从存档的肿瘤样本中提取DNA。使用侧翼引物对ras基因进行PCR扩增，并与一系列标记的等位基因特异性寡核苷酸杂交，这些寡核苷酸对应于K12,61、N12,13,61和H12,61的野生型形式以及所有替代突变组合（7种野生型，45种突变体）。
在24.2%的标本中鉴定出ras突变。在H、K和N-ras中检测到的突变均发生在密码子61处。这不是PCR或杂交假象的结果，因为在适当的对照样本中在第位置12和13处检测到了突变。
已证明突变的ras可将HPV永生化的角质形成细胞转变为致瘤状态。我们的结果表明，这些早期宫颈癌中有相当比例（24.2%）含有激活的ras。需要进一步研究以评估密码子61是否代表一部分宫颈癌中ras突变的特征性“热点”。
