Cell Cycle-Related Cyclin B1 Quantification

To obtain non-relative measures of cell proteins, purified preparations of the same proteins are used as standards in Western blots. We have previously quantified SV40 large T antigen expressed over a several fold range in different cell lines and correlated the average number of molecules to average fluorescence obtained by cytometry and determined cell cycle phase related expression by calculation from multi-parametric cytometry data. Using a modified approach, we report quantification of endogenous cyclin B1 and generation of the cell cycle time related expression profile.Recombinant cyclin B1 was purified from a baculovirus lysate using an antibody affinity column and concentrated. We created fixed cell preparations from nocodazole-treated (high cyclin B1) and serum starved (low cyclin B1) PC3 cells that were either lyophilized (for preservation) or solubilized. The lysates and purified cyclin B1 were subjected to Western blotting; the cell preparations were subjected to cytometry, and fluorescence was correlated to molecules. Three untreated cell lines (K562, HeLa, and RKO) were prepared for cytometry without lyophilization and also prepared for Western blotting. These were quantified by Western blotting and by cytometry using the standard cell preparations.The standard cell preparations had 1.5 x 10(5) to 2.5 x 10(6) molecules of cyclin B1 per cell on average (i.e., 16-fold range). The average coefficient of variation was 24%. Fluorescence varied 12-fold. The relationship between molecules/cell (Western blot) and immunofluorescence (cytometry) was linear (r(2) = 0.87). Average cyclin B1 levels for the three untreated cell lines determined by Western blotting and cytometry agreed within a factor of 2. The non-linear rise in cyclin B1 in S phase was quantified from correlated plots of cyclin B1 and DNA content. The peak levels achieved in G2 were similar despite differences in lineage, growth conditions, and rates of increase through the cell cycle (range: 1.6-2.2 x 10(6) molecules per cell).Net cyclin B1 expression begins in G1 in human somatic cells lines; increases non-linearly with variation in rates of accumulation, but peaks at similar peak values in different cell lines growing under different conditions. This suggests tight quantitative end point control

Cell density related gene expression: SV40 large T antigen levels in immortalized astrocyte lines

<p>Abstract</p> <p>Background</p> <p>Gene 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.</p> <p>Results</p> <p>Expression 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).</p> <p>Conclusions</p> <p>These 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.</p

Standard Curve.

<p>Standard Curve.</p

Comparison of Cyclin B1 Quantification.

a<p>actual value = 1.6×10⁶.</p>b<p>Used the value of Xu et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007064#pone.0007064-Xu1" target="_blank">[4]</a></p>c<p>Calculated from the published values of pmole cyclin B1/g extract and 2×10³ cells/ug lysate.</p>d<p>Measured by Kanatsu-Shinohara et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007064#pone.0007064-KanatsuShinohara1" target="_blank">[12]</a>.</p>e<p>Estimated from molecules and concentration given by Kobayashi et al. and diameter given by Chang et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007064#pone.0007064-Chang1" target="_blank">[23]</a>.</p>f<p>cyclin type in parentheses.</p>g<p>Measured by Jorgensen et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007064#pone.0007064-Jorgensen1" target="_blank">[24]</a>.</p

Comparison of MeOH fixed cells to MeOH fixed-lyophilized cells.

<p>A culture of PC3 cells was trypsinized, washed, and then divided into three equal parts. One part was solubilized for electrophoresis, one part was fixed with methanol, and the third part was fixed with methanol and then lyophilized. The methanol-fixed and lyophilized cell preparations were washed then solubilized with 23% SDS. The lanes were loaded with equal numbers of cells.</p

Cytometry and quantitative Western blots of standards.

<p>(A) Left 4 lanes are recombinant cyclin B1 at two-fold dilutions. Recombinant cyclin B1 and lysate concentrations were adjusted so that the band intensities for recombinant cyclin B1 spanned the range of those of the lysates. Trypsin inhibitor was added to each sample to bring the total protein per lane to 50 µg. (B) Lyophilized standard cell preparations from the same samples as used for Western blot were stained for cyclin B1 with GNS1-Alexa Fluor 647 (A647) and DNA (DAPI). Arrows indicate the mean cyclin B1 fluorescence. Background fluorescence was subtracted and data were plotted as a hyperlog transformation which allows plotting of negative and zero values on a linear lower section (−10 to 10) and higher values (>10) logarithmically <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007064#pone.0007064-Bagwell1" target="_blank">[22]</a>.</p

Extraction of cyclin B1 in various lysis buffers.

<p>Equal numbers of dissociated PC3 cells were solubilized in lysis buffer containing RIPA, Triton X-100 or SDS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007064#s3" target="_blank">Materials and Methods</a>) and analyzed for cyclin B1 by Western blot (A). Increasing the SDS level to 23% did not release more cyclin B1 (B). There was some variability between lanes but, based on multiple gels, it was clear that cyclin B1 was readily extracted by all methods.</p

Cell cycle expression of cyclin B1.

<p>The cytometric data for HeLa, K562, and RKO cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007064#pone-0007064-g007" target="_blank">Figure 7</a>) were gated to exclude endoreduplicated or binucleate cycling cells (4C 8C); mitotic cells after metaphase, and outliers. The purpose was to expose only the cells distributed about the two dimensional center as a function of DNA content. These data represent the most common cycling cell in the population. Arbitrary regions were set, moving through the data from early G1 through late G2+M (A–C). The mean cyclin B1 levels from each region are plotted as a function of the cell frequency in the region (D–F). The frequency data were renormalized from the first non-zero cyclin B1 point and plotted to compare the committed part of the cell cycle (cyclin B1 is detectable) for each cell line (G). In A-G, the X axis scale is 0–1, and the Y axis scale is 0–4×10⁶.</p