Macrophage-melanoma cell heterokaryons. 3. The activation of macrophage DNA synthesis. Studies with inhibitors of protein synthesis and with synchronized melanoma cells.

Dormant macrophage nuclei initiate DNA synthesis 2-3 hr after fusion of macrophages with exponentially growing melanoma cells. Cycloheximide treatment (1-5 microg/ml) of heterokaryons during the preceding lag period inhibits the initiation of macrophage DNA synthesis, in a reversible fashion. Each type of cell was also treated with streptovitacin A, an irreversible inhibitor of protein synthesis. Pretreatment of the melanoma cells (0.5-2 microg/ml), 1 hr before fusion, inhibited the induction of macrophage DNA synthesis in heterokaryons, whereas pretreatment of macrophages (1-20 microg/ml) had no effect. Melanoma cell pretreatment reduced the incorporation of leucine-(3)H into the cytoplasm and nuclei of heterokaryons, whereas macrophage pretreatment had no effect. These experiments suggested that melanoma proteins played an important role in the initiation of macrophage DNA synthesis. The relationship between the melanoma cell cycle and macrophage DNA synthesis was studied with synchronous melanoma cells. If the melanoma cells were in S phase at the time of fusion, macrophage DNA synthesis occurred 2 hr later. However, the fusion of melanoma cells in G(1) delayed macrophage DNA synthesis until the melanoma nuclei had entered S. Experiments with actinomycin and cycloheximide showed that RNA and protein, essential to achieve DNA synthesis in the macrophage nucleus, were made during late G(1) as well as S. Melanoma cells and macrophages differ in their radiolabeled acid-soluble products after incubation in thymidine-(3)H. Thymidine taken up by the macrophage remained unphosphorylated, whereas it was recovered mainly as thymidine triphosphate from melanoma cells. These findings, as well as those reported previously, suggest that the melanoma cell provides the RNA, protein, and precursors which initiate macrophage DNA synthesis. In the absence of a requirement for new macrophage RNA and protein synthesis, other changes must be responsible for the 2 hr delay in DNA synthesis. These may involve physical changes in DNA, associated with swelling, as well as the transport of melanoma products into the macrophage nucleus.