Studies on transformation of Syrian hamster cells by simian virus 40 (SV40): acquisition of oncogenicity by virus-exposed cells apparently unassociated with the viral genome.

During a study of the capacity of Syrian hamster cells derived from various organs to transform in vitro after exposure to simian virus 40 (SV40), it was found that cells from lung and liver responded differently compared to those from certain other organs. The most significant difference was the apparent absence of the viral genome in lung and liver cultures that eventually underwent transformation. Materials and Methods.-Virus: SV40, strain VA 45-54 obtained from Dr. M. R. Hilleman, was propagated in grivet monkey kidney cells, line AH-1.1 Infectivity titer of the stock virus was 106-2 TCID50/0.1 ml as determined in AH-1 cell system. Cell cultures: (a) Newborn hamster lung cells: Lungs were removed from a litter of 10 newborn animals, pooled, and trypsinized. (Throughout the article, "hamster" refers to the Syrian hamster.) Cells were grown as monolayers in 16 X 150-mm tubes. Incubation at first was at 370C in a stationary position. Thereafter, cultures were kept at 370C in a roller wheel turning at 1/4 rpm. On day 9 each one of half of the cultures was exposed to 0.1 ml undiluted stock virus, and the remainder were maintained as controls. Growth media were as previously described.2 Serial cell passages from SV40-exposed primary cultures and from controls taken at random were initiated at 1, 2, 2'/2, 3, 3'/2, and 4 months (Table 1) after addition of SV40. Subsequent passages were made every 2-3 weeks, dividing the cells in a ratio of 1: 2. At the end of the 1st, 2nd, 3rd, and 4th months after addition of virus, a culture from each group was fixed and stained, employing the collodion embedding technique. Representative cultures from subsequent passages were similarly examined. (b) Hamster embryo liver cells: Cultures were prepared from livers of 11 embryos of 2 weeks' gestation, divided into two groups, one of which was exposed to SV40 virus, and handled in the same manner as the lung cultures. Serial cell passages from SV40-exposed primary cultures and from controls were initiated at 1, 1'/2, 2, 21/2, and 3 months (Table 1) after addition of virus. Those started at 1 and 11/2 months (lines N, 0) were made from single primary cultures; those initiated at 2 and 21/2 months (lines BG, 2M) were made by pooling the cells of two tube cultures. Line 2N, initiated at 3 months, originated from a pool of cells from three primary cultures. Pooling some of the embryo liver tube cultures was adopted because passage of single cultures was made with difficulty. (c) Grivet monkey kidney cell cultures: Line AH-11 was used for assay of SV40 infectivity and tests for infective virus in culture fluids and by the Eddy-Gerber4 "overlay method." Plating efficiency: Cell suspensions were prepared from well-grown cultures in 1 ml of Puck's trypsin solution per culture, which was then added to 9 ml of growth medium. Cells were counted and aliquots of suspension diluted to contain 10' to 104 cells/ml were added to Falcon plastic dishes (60 X 15 mm) containing 3 ml of the same medium. Cultures were incubated at 370C in 5% CO2 for 10 days, fixed, stained with hematoxylin, and colonies counted. Tests for infective SV4O: (1) Undiluted culture fluid was centrifuged and the supernatant added in aliquots of 0.5 ml to cultures of AH-1 cells. (2) By a slightly modified Eddy-Gerber "overlay method," 0.2 ml of a suspension of trypsinized cells was overlaid on AH-1 cultures. At the end of 2, 4, and occasionally 6 weeks, centrifuged supernatant fluid from the latter was added to fresh cultures of AH-1 cells, which were examined biweekly for cytopathogenic effect and at the end of 6 weeks were fixed and stained. Immunoftuorescence technique: The indirect test for "cellular" or "tumor" antigen in SV40-