Student Theses and Dissertations
Date of Award
1971
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Zanvil Cohn
Keywords
macrophage-melanoma fusion, heterokaryon activation, DNA synthesis induction, melanoma-derived RNA/protein, membrane receptor masking, gene expression regulation
Abstract
Mouse peritoneal macrophages, which do not synthesize DNA in vitro, were fused with a strain of mouse melanoma cells which proliferates rapidly in vitro. The plasma membrane of the macrophage has specific receptors which enable the cell to ingest antibody-coated sheep red cells and is also rich in a divalent cation dependent adenosine triphosphatase (ATPase) activity. The melanoma cell lacks these macrophage membrane markers. High yields of mouse macrophage-melanoma cell heterokaryons and macrophage-macrophage homokaryons were obtained through the Sendai virus-induced fusion of cells spread on a glass surface. After fusion there was a striking reorganization of cellular architecture by means of a colcemid-sensitive process. Heterokaryons were isolated through the use of differential trypsinization and many underwent division to form melanoma-like hybrids. The selective uptake of dextran sulfate by macrophages served as a useful cytoplasmic marker in identifying hybrids. Many characteristic macrophage properties were altered in the heterokaryons. Within an hour of fusion macrophage nuclei became swollen, nucleoli were more prominent and increased nuclear RNA synthesis occurred. Two - three hours after fusion a wave of DNA synthesis took place in the previously dormant macrophage nuclei. DNA synthesis was induced in macrophage nuclei irrespective of the number of macrophage nuclei present per melanoma nucleus in each heterokaryon. Fifty to 80% of macrophage nuclei initiated DNA synthesis in the three - seven hour period after fusion. The activation of most 11 - 12 day chick red cell nuclei in melanoma cytoplasm took longer than ten hours. 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 one - four hours before the DNA and was unlikely to be ribosomal RNA since it was insensitive to < 0.1 μg/ml actinomycin. Melanoma cells and macrophages were treated before fusion with actinomycin and bromotubercidin to bring about a more selective inhibition of RNA synthesis. Macrophages pretreated for one hour with 5 μg/ml actinomycin showed less than 20% of control RNA synthesis in the first four hours after fusion, but a normal activation of macrophage DNA synthesis. Pretreatment of melanoma cells for three - seven hours with 5 μg/ml bromotubercidin, a reversible inhibitor of RNA synthesis, prevented macrophage DNA synthesis without affecting macrophage RNA synthesis in the heterokaryon (81% of control). These studies showed that only melanoma 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 is activated in the heterokaryon it becomes less sensitive to the action of actinomycin. Cycloheximide treatment (1-5 μg/ml) of heterokaryons during the preceding lag period inhibited the initiation of macrophage DNA synthesis in a reversible fashion. Each type of cell was also treated with streptovitac in A, an irreversible inhibitor of protein synthesis. Pretreatment of the melanoma cells (1-2 μg/ml), one hour before fusion, inhibited the induction of macrophage DNA synthesis in heterokaryons, whereas pretreatment of macrophages had no effect. Melanoma cell pretreatment reduced the incorporation of 3H leucine 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 two hours later. However, the fusion of melanoma cells in G1 delayed macrophage DNA synthesis until the melanoma cells 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 G1 as well as S. Melanoma cells and macrophages differ in their radio-labeled acid-soluble products after incubation in 3H thymidine. Thymidine taken up by the macrophage remained unphosphorylated, whereas it was recovered mainly as thymidine triphosphate from melanoma cells. These findings 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 two - three hour 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. The fate of the macrophage membrane markers was examined in both heterokaryons and homokaryons. Macrophage homokaryons continued to exhibit active phagocytosis of sensitized erythrocytes. The phagocytic receptor could be detected in heterokaryons shortly after fusion, but was progressively lost, at an exponential rate, over the next 12 - 24 hours. Another macrophage surface marker, the ATPase, could be demonstrated histochemically on heterokaryons. Shortly after fusion, it was present in discrete regions, but it became more diffuse and disappeared within a day. Exposure of heterokaryons to trypsin (1-100 μg/ml/30'/37°C) results in the reappearance of initial receptor activity and the unmasking of the surface receptor. This property is again lost upon subsequent cultivation. The masking process takes place when cells are cultivated in the absence of IgG so that the adsorption of antibody from the medium is not responsible for this phenomenon. Inhibition of heterokaryon protein synthesis preserves phagocytic activity in a reversible fashion and prevents the masking of macrophage receptors. Inhibition of melanoma RNA synthesis before fusion is also able to block subsequent masking, but is ineffective if delayed until after fusion. U-v irradiation of the melanoma cell before fusion prevents subsequent masking, whereas similar treatment of the macrophage has no effect. Antibody coated red cells attach diffusely to the surface of unmasked heterokaryons, suggesting that the macrophage receptor has become distributed over the whole cell surface. The macrophage receptor undergoes a change in function soon after fusion with a melanoma cell, since a higher concentration of antiserum is necessary to bind red cells to the heterokaryon. This change is independent of heterokaryon protein synthesis. These results suggest that heterokaryon formation causes an early spreading and mixing process of some membrane components, as well as a slower masking process. Melanoma RNA synthesis results in the production of membrane protein which is responsible for the masking phenomenon, thus illustrating a novel mechanism for altering the expression of plasma membrane properties. The question of macrophage receptor synthesis was investigated in DBA/2 mouse macrophage x mouse LMTK- cell hybrids. Three clones and one mass culture were isolated by their ability to grow in HAT selection medium. These hybrids retained 85 - 100% of the sum of their parental chromosomes and expressed genes derived from both parent cells, including glucose phosphate isomerase isozymes and H-2 antigens. The hybrids displayed ATPase activity intermediate between that of the macrophage and L cells. The macrophage-specific receptors for antibody coated red cells or complement could not be demonstrated on hybrid cells. It is unlikely that the selective absence of these receptors was due to loss of the appropriate genes.
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Recommended Citation
Gordon, Siamon, "Nuclear and Plasma Membrane Properties of Macrophage Heterokaryons and Hybrids" (1971). Student Theses and Dissertations. 547.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/547
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy