Topoisomerase activities have heen measured in nuclear extracts of concanavalin A-stimulated lymphocytes. In parallel with the wave of DNA synthesis, type II topoisomerase activity was considerably increased. After 72 h treatment, this activity was stimulated approx. 20-fold over the activity in untreated cells. In contrast, type I topoisomerase was poorly stimulated after 24 h treatment, and 4-5-fold after 72 h. These findings, together with our previous results on regenerating rat liver, suggest a major role of topoisomerase II in DNA replication.
Mitogenic agent DNA replication
The actual dilemma in studying the binding and triggering capacity of IgE from allergic patients is the lack of cultured basophils or mast cell analogs of human origin. Human IgE binds with exquisite species specificity to the high affinity IgE receptor (FcεRI) expressed on the surface of these cells. In rodents this receptor has been characterized as a tetrameric plasma membrane protein composed of an IgE-binding α chain, a β chain and two disulfide-linked γ chains. In order to establish a cell line expressing the α chain of human FcεRI which can be triggered with IgE from human patients and specific allergen, we transfected the cDNA coding for the human α subunit into rat basophilic leukemia cells. The resulting transfectants express the human α chain on the cell surface in the form of a hybrid complex associated with endogenous rat γ chains. After sensitization with human IgE from mite-specific patients, the transfectant produces a calcium response upon incubation with allergen. The established cell line can be used as a model system to study the mechanism of mast cell triggering through IgE from allergic patients.
Etoposide, a nonintercalative antitumor drug, is known to inhibit topoisomerase II. Its effects have been tested in concanavalin A stimulated splenocytes, a system of cell proliferation in which topoisomerase II is induced. The primary effect of etoposide was a strong inhibition of DNA synthesis and the production of reversible DNA breaks, presumably associated with topoisomerase II. However, prolonged (20 h) contact with the drug resulted in a secondary fragmentation by irreversible double-strand breaks that yielded unusually small DNA fragments. Surprisingly, the same effect was obtained with novobiocin, which does not produce topoisomerase II associated DNA breaks. Moreover, long-term treatment with camptothecin, a specific inhibitor of topoisomerase I which is known to induce single-strand breaks in vitro and in vivo, also produced double-strand breaks and DNA fragmentation into small pieces. These findings suggest that prolonged treatment of proliferating splenocytes by etoposide and other topoisomerase inhibitors induced DNA fragmentation by a mechanism that does not directly involve topoisomerases.
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