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Effects of Arabidopsis Ick1-Mediated Cell-Cycle Arrest on the Physiology and Recombinant Protein Production in Tobacco Cell Suspension Cultures

Wei Wen Su, T. Venkatappa, K. Nickolov, and B. Liu. Molecular Biosciences & Bioengineering, University of Hawaii, 1955 Eastwest Rd., Ag Sci 218, Honolulu, HI 96822

Controlled-proliferation by genetically arresting cell cycle progression has been shown to increase the production of heterologous recombinant proteins in mammalian cell cultures. In this study, we examine the applicability of such approach in plant cell cultures. We constructed transgenic Nicotiana tabacum cell lines that concomitantly carry a cyclin dependent kinase inhibitor gene (Arabidopsis thaliana ICK1, controlled by an estrogen-inducible XVE promoter) and a reporter gene (GFP, driven by the constitutive CaMV35S promoter). Flow cytometry analysis of the ICK-1 expressing cells revealed a high degree of cell cycle arrest at G1/S. Reduced CDKa kinase activity was also noted in these cells. The apparent cell proliferation and glucose consumption rates of the ICK1-expressing cell suspension culture were lower than those of the proliferation-competent control cell lines, although proliferation was not completely arrested. This may be explained by the observation with a stably transformed tobacco cell line expressing a GFP driven by the XVE promoter, in which only a portion of the cell population (less than 50%) displayed fluorescence upon induction. Accompanying the apparent lower growth rate was the observation of prolonged growth (i.e. delay in culture decay). These results support the effectiveness of inducible ICK-1 expression on controlled proliferation of culture plant cells. However, unlike in the mammalian systems, the recombinant reporter protein production was not improved as a result of ICK-1 expression. Cellular GFP transcript and protein levels (as a percentage of total RNA and protein level, respectively) remained unchanged upon ICK1 induction.