Alterations Of Oncogenes In Human Fetal Esophageal Epithelium Induced By N-Methylbenzylnitrosamine (NMBzA)

Esophageal cancer (EC) is the second most common cancer in China. In Linxian county, a high incidence area, it is the leading cause of cancer deaths. Previous studies have shown that N — nitroso compounds (NOC) are the prevailing risk factors. N—methylbenzylnitrosamine (NMBzA) is a naturally occurring carcinogen, and exposure to this nitrosamine has been associated with increased incidence of EC in Linxian county. In support of this contention, our laboratory has demonstrated that EC could be induced in human fetus by NMBzA in vitro. However, the molecular machanisms of esophageal carcinogenesis is poorly understood at present. The purpose of this thesis is to determine whether activation of oncogene(s) and inactivation of antioncogene(s) are involved in the induction of malignant transformation of human fetal esophageal epithelium (HFE) by NMBzA. The results of our studies are summaried as follows:1. Amplification and overexpression of EGFr gene were found in HFE treated with NMBzA for 24 hours as shown by Southern blot assay and immunohistochemistry, indicating that activation of EGFr gene might be an initiating event in esophageal carcinogenesis induced by NMBzA.2. Papillary hyperplasia was induced in HFEs cultured with NMBzA for 1 to 3 weeks. Using molecular hybridization, amplification of c—myc and INT—2 gene was found in HFEs induced for 1 week and 3 weeks, respectively. These results suggest that activations of c —myc and INT—2 gene might be associated with clonal proliferation and expansion of the initiated cells.3. Hybridized with specific p53 and Rb gene cDNA probe, deletions of the p53 and Rb genes were found in human fetal esophageal carcinomas induced by NMBzA. Overexpression of p53 protein in human fetal esophageal carcinomas detected immunohistochemically indicated that p53 gene mutation (s) might have be occurred. It has been shown that overexpression of p53 protein in human cancer is the result of p53 gene mutations. However, we could not detect any deletion and/or overexpression of Rb and p53 gene in HFE treated in vitro for 24 hours to 3 weeks with NMBzA. These results suggest that inactivation of Rb and p53 antioncogenes might be involved but a late event in esophageal carcinogenesis.4. The fetal esophageal epithelium explants were treated with NMBzA for 24 hours, then its high molecular weight DNA extracted, and used to transfect Rat—1 cells by the DNA —Ca₃ (PO4)₂ technique. The transforming efficiency was 0. 068 foci/ug DNA. In the secondary transfection, the transformation efficiency increased to 0. 18 foci/ug DNA. The transformed cells were anchorage independent in 0.3% soft agar, and developed into tumor in balb/c nude mice. Molecular hybridization and PCR amplification proved that in the genome of the transformants there were Alu sequences and H—ras gene, but no evidence of codon 12 point mutation of the H — ras gene by PCR — RFLP analysis. Using immunohistochemical methods, we did not find O6 — MedG adducts in HFE induced for 24 to 72 hours.5. DNA extracted from HFEs treated with NMBzA was analyzed by Southern blot assay. Deletion of H —ras gene was detected in HFE treated for 3 weeks and in EC induced by NMBzA, indicating that loss of H —ras gene may play a role in the pathogenesis of NMBzA — induced EC. Combined with other relevent...