Effect of prolonged cultivation of SV40-transformed mouse cells in bromide-oxyuridine or pretreatment with mitomycin C on rescue of SV40.

Abstract

AbstractA series of clonal lines (mKS‐U) of mouse kidney cells transformed by UV‐irradiated SV40 have been propagated serially in medium containing 5‐bromodeoxyuridine (dBU). Most of the mKS‐U lines became resistant to dBU during serial passage and became deficient in thymidine (dT) kinase activity. The dT kinase‐deficient mKS‐U lines were unable to grow in medium containing HATG (10−4 M hypoxanthine, 10−5 M aminopterin, 4 × 10−5 M thymidine, and 10−5 M glycine; Littlefield, 1965) because they were unable to utilize exogenous thymidine as a source of thymidylate when de novo synthesis of thymidylate was blocked by aminopterin. Growth in dBU medium did not increase the buoyant density of the DNA of mKS‐U lines deficient in dT kinase activity, demonstrating that dBU was not incorporated into the DNA of the cells. These observations show that a heritable change had occurred in the dBU‐grown mKS‐U cell lines. However, two dBU‐adapted cell lines, mKS‐U1 (BU) and mKS‐U2 (BU), did retain appreciable dT kinase activity even after 97 and 98 passages in dBU. The DNAs from mKS‐U1 (BU) and mKS‐U2 (BU) grown in dBU had buoyant densities greater than those of the DNAs from mouse cells grown in medium lacking dBU and the mKS‐U1 (BU) and mKS‐U2 (BU) lines were able to grow normally in HATG medium.Serial passage of poor, average, or good yielder transformed lines in dBU did not significantly change the frequency of induction of SV40 in fusion experiments with African green monkey kidney (CV‐1) cells. However, the SV40 rescued in fusion experiments with two poor yielders after serial passage in dBU tended to produce larger plaques than those rescued prior to passage in dBU. Serial passage in dBU did not permit rescue of SV40 from heterokaryons of the rare yielders or non‐yielders.The SV40 antigen was present in all the cell lines, even after prolonged cultivation in dBU, indicating that the SV40 genome was retained.Pretreatment of mKS‐U lines with mitomycin C prior to fusion with CV‐1 cells did not alter the virus yield from a good yielder, mKS‐U13, after fusion with CV‐1, and did not permit rescue of SV40 from rare or non‐yielder lines.