DMBA 도포로 유발된 Hamster 협낭의 구강암조직에서 in Situ Reverse Transcriptase(RT) PCR 기법에 의한 EGFR mRNA의 발현

Abstract

Epidermal growth factor receptor(EGFR) mRNA was assessed in 7, 12-dimethylbenz(a) anthracene(DMBA)-induced squamous cell carcinomas(SCC) in the hamster buccal pouch model to elucidate the role and timing of histologic changes and differentiation during carcinogenesis. In situ reverse-transcriptase polymerase chain reaction was used to identify the EGFR. DMBA(0.5%) in heavy mineral oil was applied to the right buccal pouch 3 times per week for up to 16 weeks. Hyperplasia was detected by histologic analysis at 4 weeks, dysplasia with or without papillomatous changes at 8 weeks, and SCC at 16 weeks. Paraffin embedded sections of the tumors were used for EGFR mRNA and immunohistochemical determinations. EGFR cDNA was synthesized in situ by reverse transcription using an EGFR-specific oligonucleotide primer. In situ PCR amplification in the presence of digoxigenin-11-dUTP and subsequent binding with an antidigoxigenin antibody conjugated to alkaline phosphatase allowed direct visualization. EGFR mRNA was localized in the nuclei of the basal cell layer of normal squamous epithelium but is expanded to the superficial squamous cell layer as well as the basal cell layer in hyperplasia and dysplasia and is diffusely expressed in squamous cell carcinoma. EGFR protein, detected by immunostaining using a monoclonal antibody, was expressed mainly in the cytoplasm of superficial squamous cell layers(not in the basal cell layer) in normal squamous epithelium. It increased gradually in level and amount through the stages of hyperplasia and dysplasia to invasive squamous cell carcinoma. These results suggest that the biological markers EGFR mRNA and EGFR protein may be used for assessing intermediate end points in tests of various chemopreventive agents on oral carcinogenesis in the hamster buccal pouch model as well as in human clinical trials.