The fate of the DNA of adenovirus type 12 in baby hamster kidney cells.

There is evidence that the viral genome, or part of it, persists and continues to be transcribed' in cells derived from tumors induced by human adenovirus type 12 (ad 12) and in cells transformed in vitro by ad 12. It is not clear whether the viral genome is covalently linked to cellular DNA (integration) or replicates as free DNA in phase with cellular DNA. In the studies reported here, it is proposed that the decisive events leading to transformation are most likely to occur soon after infection of cells susceptible to transformation by ad 12. A system was developed that permitted an investigation of the fate of the DNA of ad 12 in such cells. Baby hamster kidney cells (BHK21),2 which can be transformed by ad 12 in vitro,3 were chosen for these studies. In this communication, evidence will be presented for the first time that the DNA of ad 12, or part of it, can become linked to the DNA of BHK21 cells. The significance of these findings for the phenomenon of transformation remains to be elucidated. A preliminary report of this work has been given.4 Materials and Methods. -Cell culture media: Spinner medium is Eagle's medium' without CaC12 enriched with each of the nonessential amino acids (100 MAM) and 10% calf serum. ETC is reinforced Eagle's medium6 with 10% tryptose phosphate broth (TPB) and 10% calf serum. Solutions: PBS is phosphate-buffered saline;7 TE is 0.01 Al Tris, pH 7.2, 0.001 Al ethylenediaminetetraacetate (EDTA); and STE is 0.5% sodium lauryl sulfate, 0 05 Al Tris, pH 7.5, and 0.01 M EDTA. Cells and virus: KB cells8 (CCL17) were obtained from the American Type Culture Collection. BHK21 cells, clone 13, were originally isolated by Stoker and Macpherson2 and were given to us by Dr. Carabazzo of the Rockefeller Foundation Virus Laboratory. Primary human embryonic kidney (HEK) cells were purchased from Microbiological Associates, Inc. Seed virus preparations of human adenovirus type 12, strain Huie, were supplied by the American Type Culture Collection and by Dr. Wallace Rowe of the National Institute of Allergy and Infectious Diseases. Chemicals: CsCl was obtained from the Harshaw Chemical Co. as optical-grade powder. Pronase, B-grade, and 5-bromodeoxyuridine (5-BUdR) were obtained from Calbiochem. , Inc. Dr. A. Kornberg of Stanford University supplied dAT-copolymer. All other chemicals used were of analytical grade. Radioisotopes: Thymidine-methyl-H3 (15-17 c/mmole), thymidine-2-C"4 (30 mc/ mmole), and C14-amino acids mixture (uniformly labeled) were purchased from the New England Nuclear Corp.; thymidine-6-H3 (17-22 c/mmole) was purchased from NuclearChicago, and 5-BUdR-6-H3 (2.5 c/mmole) from Schwarz BioResearch, Inc. Plaque assay of ad 12: The procedure of Strohl et al.3 was followed with minor modifications. Virus growth and purification: Ad 12 was grown in KB cells in suspension cultures in Spinner medium and was purified by a modification' of the procedure of Green and Pifia. '0 All virus preparations used in this study were purified by three cycles of equilibrium sedimentation in CsCl. They were found to be free of contaminating adenoassociated virus by ultracentrifugal and electron microscopical examinations. The final