Macrophage-melanocyte Heterokaryons. II. The Activation of Macrophage DNA Synthesis. Studies with Inhibitors of RNA Synthesis

Mouse peritoneal macrophages, which do not synthesize DNA in vitro, were fused with melanocytes, a mouse cell strain which proliferates rapidly in vitro. DNA synthesis was induced in macrophage nuclei 2-3 hr after fusion and occurred irrespective of the number of macrophage nuclei present per melanocyte nucleus in each heterokaryon. 50-80% of macrophage nuclei initiated DNA synthesis in the 3-7 hr period after fusion. The activation of most 11-12-day chick red cell nuclei in melanocyte cytoplasm took longer than 10 hr. The lag before DNA synthesis may reflect the heterochromatin content of each nucleus. Studies with actinomycin showed that heterokaryon RNA synthesis was essential for subsequent macrophage DNA synthesis. This RNA was synthesized 1-4 hr before the DNA and was unlikely to be ribosomal RNA, since it was insensitive to <0.1 microg/ml actinomycin. Melanocytes and macrophages were treated before fusion with actinomycin and bromotubercidin to bring about a more selective inhibition of RNA synthesis. Macrophages pretreated for 1 hr with 5 microg/ml of actinomycin showed less than 20% of control RNA synthesis in the first 4 hr after fusion, but a normal activation of macrophage DNA synthesis. Pretreatment of melanocytes for 3-7 hr with 5 microg/ml bromotubercidin, a reversible inhibitor of RNA synthesis, prevented macrophage DNA synthesis without affecting macrophage RNA synthesis in the heterokaryons (81% of control). These studies showed that only melanocyte RNA synthesis was essential for the production of macrophage DNA. The exposure of one cell partner to actinomycin before fusion caused cross-toxicity of the untreated nucleus after fusion. Bromotubercidin, an adenosine analogue which is incorporated into RNA, did not give rise to such cross-toxicity after fusion. Once the macrophage nucleus becomes activated in the heterokaryon it becomes less sensitive to the action of actinomycin.