Reversible “Transformation” of Bromodeoxyuridine-Dependent Cells by Bromodeoxyuridine

Abstract

The growth characteristics of a bromodeoxyuridine-dependent cell line that was derived from a non-contact-inhibited Syrian hamster melanoma line were studied. The dependent cells require high concentrations of bromodeoxyuridine not only for optimal growth but also for the maintenance of the non-contact-inhibited state. When grown in the absence of bromodeoxyuridine, the dependent cells become contact-inhibited. The transition to the contact-inhibited state is reversed when bromodeoxyuridine is added back to the medium, if the bromodeoxyuridine is incorporated into dividing cells. The effects of bromodeoxyuridine on growth rate and on contact inhibition are separable.We recently described a new mutation in mammalian cellsbromodeoxyuridine dependence (1). The mutant cells require the drug 5-bromodeoxyuridine (BrdU) for optimal growth, and the concentration of BrdU at which these cells grow most rapidly is toxic to most other cells. In the absence of BrdU, the mutant cells continue to multiply but at a significantly reduced rate. The requirement for BrdU in these cells is specific, not being satisfied by thymidine or related analogues such as iododeoxyuridine. The mutant cells incorporate BrdU into nuclear DNA, replacing approximately 50% of the thymidine residues with BrdU. In the presence of BrdU plus inhibitors of thymidine biosynthesis, the cells grow with essentially 100% of the thymidine replaced by BrdU (2).The BrdU-dependent cells were derived from a highly malignant Syrian hamster melanoma cell line. Like other malignant cells in culture, the melanoma cells are not contactinhibited, i.e., they do not cease growing when they have formed a confluent monolayer. In the presence of BrdU, the BrdU-dependent cells are similarly not contact-inhibited. However, in the absence of BrdU, the dependent cells become contact-inhibited. This change in the pattern of growth is of special interest because contact inhibition is a characteristic generally associated with nonmalignant cells. (The mechanisms of contact inhibition are not understood, and the role of cell contact per se remains to be demonstrated.) This paper describes the requirement of BrdU for the maintenance of the non-contact-inhibited state in the BrdU-dependent cells. MATERIALS AND METHODSCell Lines and Growth Media. The isolation of the BrdUdependent cell line B4 from the Syrian hamster melanoma line RPMI 3460 was described previously (1). The 3460 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal-calf serum (E medium). The B4 cells were maintained in E medium containing 0.1 mM BrdU (E-B medium). A revertant cell line, able to grow well in BrdU but no longer requiring it, was isolated from B4. The B4 cells were first cloned in E medium, and a clone of rapidly growing cells, called B4-E18, was isolated. The B4-E18 cells were then cloned in E-B medium, and a subclone, B4-E18-B2, which grew well in the presence of BrdU was isolated. All of the cell lines were protected from exposure to light of wavelengths below 550 n...