Visualization of c-Ki-ras-2 oncogene sequences in human pancreas, a chemically induced transplantable carcinoma, and carcinomas of pancreas by in situ hybridization.

c-Ki-ras-2 sequences were visualized in paraffin-embedded sections from normal fetal and adult human pancreases, a chemically induced transplantable human pancreas carcinoma (PT-1) and three carcinomas of pancreas by in situ hybridization technique. A biotinylated 1-kilobase-pair (kb) EcoRI fragment of pHiHi3 DNA was used as probe and the oncogene was visualized as one or two large grains of reaction products produced by streptavidin-peroxidase complex and diaminobenzidine tetrachloride in more than 9% of normal pancreas nuclei. Its amplification in the chemically induced cell line was detected as one or more large grains in 72% of the nuclei and numerous cytoplasmic grains. The detection of oncogene in normal pancreases and its amplification in PT-1 cells was validated by Southern analysis of EcoRI digests of genomic DNA extracted from normal pancreases and PT-1 cell line. The oncogene was also demonstrated to be equally amplified in two adenocarcinomas and one undifferentiated carcinoma of human pancreas by in situ hybridization.

Methylnitrosourea-induced carcinoma in organ-cultured fetal human pancreas.

Explants from 12- to 14-week-old human fetal pancreases were organ cultured in a chemically defined medium and cultured for up to 12 months in the presence or absence of methylnitrosourea (MNU). Differentiation of the exocrine pancreas occurred in vitro, and explants cultured in the absence of MNU for 4 weeks or longer revealed normal acinar structures with zymogen granules. Ducts and ductules also developed normally. The undifferentiated tubular structures of the 12- to 14-week fetal pancreas expressed neither ductal nor acinar cell markers. Acinar cell surface marker appeared first after 2 weeks of culture, and the development of centroacinar and ductal cell markers followed 2 to 4 weeks later. MNU-treated explants showed minimal degeneration and necrosis. MNU caused early loss of apical cytoplasm and zymogen granules in acinar cells, resulting in dilation of acinar lumens, concomitant proliferation of cells bearing duct cell markers, and ductal hyperplasia. Enhanced foci of proliferation and carcinoma developed within 3 and 5 months of treatment, respectively. Cells derived from 4- to 5-month MNU-treated explants were tumorigenic in nude mice. Tumor cells revealed a human karyotype and expressed duct cell surface markers.