Estimation of kinetic cell-cycle-related gene expression in G1 and G2 phases from immunofluorescence flow cytometry data

Background: Flow cytometry of immunofluorescence and DNA content provides m easures of cell-cycle-related gene expression (protein and/or epitope level s) for asynchronously growing cells. From these data, time-related expressi on through S phase can be directly measured. However, for G1, G2, and M pha ses, this information is unavailable. We present an objective method to mod el G1 and G2 kinetic expression from an estimate of a minimum biological un it of positive immunofluorescence derived from the distribution of specific immunofluorescence of mitotic cells. Methods: DU 145 cells were stained for DNA, cyclin B1, and a mitotic marker (p105) and analyzed by flow cytometry. The cyclin B1 immunofluorescence (B 1) distribution of p105-positive cells was used to model the B1 distributio n of G2 and G1 cells. The G1/S and S/G2 interface measurements were used to calculate expression in S phase and test the validity of the approach. Results: B1 at S/G2 closely matched the earliest modeled estimate of B1 in G2. B1 increased linearly through G1 and S but exponentially through G2; mi totic levels were equivalent to the highest G2 levels. G1 modeling of B1 wa s less certain than that of G2 due to low levels of expression but demonstr ated general feasibility. Conclusions: By this method, the upper and lower bounds of cyclin B1 expres sion could be estimated and kinetic expression through G1, G2, and M modele d. Together with direct measurements in S phase, expression of B1 throughou t the entire cell cycle of DU 145 cells could be modeled. The method should be generally applicable given model-specific assumptions. (C) 1999 Wiley L iss,mc.