Identification of H, K, and N-ras point mutations in Stage IB cervical carcinoma

Abstract

Objective: The ras oncogenes, Harvey (H), Kitsten (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. Methods: 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, N12,13,16, and H12,61, as well as to all combinations of substitution mutations (7 wild-type, 45 mutants). Results: 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. Conclusions: 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.