demecolcine and Fibrosarcoma

The relatively low concentrations of retroviral vectors produced by most packaging cells requires the optimization and intensification of their production to make a commercially viable product for gene therapy. While a number of reports exist concerning target cell-cycle effects on retroviral vector infection efficiency, no studies have been reported on the effects of packaging cell cycle on vector production. We have studied the effect of proliferation of the human packaging cell line, FLYRD18, on vector production. In addition, the titer levels of vector produced by cells in each phase of the cell cycle were compared. Numerous studies suggested progression of the cells through the cell cycle to be essential for vector production. However, vector release was found not to be predominant in any particular phase of the cell cycle. These findings indicate that packaging cell proliferation is important for optimal virus production and that arrest of the cells in any particular phase of the cell cycle affords no benefits in retroviral vector production. In contrast to previous reports (using other cell lines), we observed no temporary inhibition of cell cycle progression after detachment of cells from their substratum and that virus production occurred immediately after re-plating of the cells. The findings in this report are important for determining the optimal culture conditions for vector production by packaging cells in vitro.

Topics: Cell Count; Cell Culture Techniques; Cell Cycle; Cell Division; Cell Line; Cell Separation; Culture Media; Demecolcine; Fibrosarcoma; Flow Cytometry; Genetic Vectors; Humans; Retroviridae; Thymidine; Tumor Cells, Cultured; Virus Replication

Induction of polyploidization by colcemid in cultured fibrosarcoma cells (Meth-A cells) was examined. Activators of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA) and ATP, inhibited colcemid-induced polyploidization, but not colcemid-induced cell proliferation cessation. These findings suggest that a down-regulation of PKC activity results in checkpoint "dysfunction" which induces polyploidization and that inhibition of polyploidization induction by PMA and ATP is not a result of the inhibition of colcemid-induced depolymerization of tubulin.

Topics: Adenosine Triphosphate; Animals; Cell Division; Demecolcine; Enzyme Activation; Fibrosarcoma; Methylcholanthrene; Mice; Mice, Inbred BALB C; Mitosis; Neoplasms, Experimental; Polyploidy; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured