Mass Enucleation of Hela 1437 Cells via Treatment with Cytochalasin B and Centrifugation
McCann, Lynn A.
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One virus currently under examination at Wayne State University Medical School is the respiratory syncytial virus (BSV) which is responsible for bronchial infections in children (Levine, 1974). In the laboratory, HeLa cells (isolated and cultured from a biopsy on a carcinoma of the neck of the uterus) are used as host cells for the replication of the RSV (Levine, 1974). The laboratory, in which I did my SIP is engaged in a study of the synthesis of RSV-RNA and protein by the infected cell. Because the quantity of viral RNA and protein synthesized in the infected cell may be only a small proportion of the total RNA and protein made by the infected cell, an attempt is being made to find a method to reduce the rate of cellular RNA and protein synthesis that will not reduce the rate of viral RNA and protein synthesis (Levine, 1974). One method currently under study is the removal of the host cell nucleus by treatment of the cell with cytochalasin B (cyto B) followed by centrifugation, because removal of the nucleus stops cellular RNA synthesis, and eventually leads to a decrease in the rate of cellular protein synthesis (Levine, 1974). Combining the enucleating-tendencies of cyto B with suitable rates and periods of centrifugation appears to be an adequate method of producing large numbers of intact enucleate cells. All the sources previously cited agree that there is a noticeable difference in the morphology of the cells after centrifugation (in general, a tendency toward a more elongate shape). However, these sources also agree that incubation for 15 minutes to 2 hours in fresh nutrient medium will return the cells to their original morphology. Therefore, treatment with cytochalasin B followed by centrifugation appears to be a suitable method by which to enucleate cell cultures. It was hoped that removal of the nucleus from the HeLa cell would reduce the rate of synthesis of cellular RNA and protein but still permit the replication of RSV. If this procedure was successful, enucleated cells infected with RSV would incorporate radiolabelled precursors of RNA and protein only into viral RNA and protein. The purpose of the procedures that follow, then, was to utilize existing techniques for the enucleation of mammalian cells with cytocha1asin B, and to determine the best procedure for the mass enucleation of HeLa cells in culture.