Abstract
BackgroundGene expression is affected by population density. Cell density is a potent negative regulator of cell cycle time during exponential growth. Here, we asked whether SV40 large T antigen (Tag) levels, driven by two different promoters, changed in a predictable and regular manner during exponential growth in clonal astrocyte cell lines, immortalized and dependent on Tag.ResultsExpression and cell cycle phase fractions were measured and correlated using flow cytometry. T antigen levels did not change or increased during exponential growth as a function of the G1 fraction and increasing cell density when Tag was transcribed from the Moloney Murine Leukemia virus (MoMuLV) long terminal repeat (LTR). When an Rb-binding mutant T antigen transcribed from the LTR was tested, levels decreased. When transcribed from the herpes thymidine kinase promoter, Tag levels decreased. The directions of change and the rates of change in Tag expression were unrelated to the average T antigen levels (i.e., the expression potential).ConclusionsThese data show that Tag expression potential in these lines varies depending on the vector and clonal variation, but that the observed level depends on cell density and cell cycle transit time. The hypothetical terms, expression at zero cell density and expression at minimum G1 phase fraction, were introduced to simplify measures of expression potential.
Highlights
Gene expression is affected by population density
The astrocyte lineage of the tkT lines was confirmed by the presence of the astrocyte-specific intermediate filament protein, GFAP [14] (Figure 1)
Lysates from equal numbers of cells were loaded on the electrophoresis gel with the exception of P0-13D tkT#13, which expressed the lowest levels of T antigen (Tag)
Summary
Gene expression is affected by population density. Cell density is a potent negative regulator of cell cycle time during exponential growth. We have explored the relationship of SV40 large T antigen (Tag) expression as a function of cell density and cell cycle duration in clonal populations of Tag-immortalized mouse astrocytes. These cells depend on expression of T antigen for viability. Expression of Tag has profound effects on the cell cycle, significantly reducing cell doubling time and increasing saturation density [4,5,6]. These direct effects of Tag result from binding and (page number not for citation purposes)
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