Timing the changes of cyclin E cell content in G1 in exponentially growing cells.
We present a method for measuring the content of immunocytochemically detected proteins in individual cells progressing through G(1) phase and its application in the analysis of cyclin E levels. The sequence of G(1) events is tracked under unaltered cycling conditions, in a cell line in the phase of balanced growth in vitro, to avoid the pitfalls of synchronization. Cells were pulse-labeled with BrdUrd and analyzed sequentially by multiparameter flow cytometry, focusing on the subpopulation of labeled cells progressively entering G(1). We use the time-from-birth ("age") of individual cells to track their position inside G(1). Using the average content of cyclin E in the whole population of G(1) cells as the internal reference for each sample, we analyzed the time course of the frequency histograms of cyclin E content within BrdUrd-labeled G(1) cells by exploiting the properties of the age distributions of asynchronous populations. This way we could calculate the average cyclin E content of cells in each age cohort. Cyclin E values were low until age 3 h, after which they rose gradually, reaching six times the value of newborn cells at the end of G(1).
Tomasoni
Daniela
D
Biophysics Unit, Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy.
Lupi
Monica
M
Brikci
Fadia Bekkal
FB
Ubezio
Paolo
P
D001973:Bromodeoxyuridine / Q000032:analysis; D002455:Cell Division; D002460:Cell Line; D019927:Cyclin E / Q000032:analysis*; D004247:DNA / Q000032:analysis; D005240:Feasibility Studies; D005434:Flow Cytometry; D016193:G1 Phase; D006801:Humans; D007700:Kinetics; D008722:Methods; D008954:Models, Biological; D013997:Time Factors
D019927:Cyclin E; D004247:DNA; D001973:Bromodeoxyuridine