Membrane-elution analysis of content of cyclins A, B1, and E during the unperturbed mammalian cell cycle

Background: Problems with whole-culture synchronization methods for the study of the cell cycle have led to the need for an analysis of protein content during the cell cycle of cells that have not been starved or inhibited. The membrane-elution method is a method that allows the study of the cell cycle by producing a culture of unperturbed, synchronized cells. Results: The Helmstetter membrane-elution method for the continuous production of newborn, unperturbed, mammalian cells has been enhanced so that the collection of cells of different cell cycle ages is automated, reproducible, and relatively inexpensive. We have applied the automated membrane-elution method to the analysis of cyclin content during the cell cycle. Cyclin E protein was invariant during the cell cycle. Cyclins B1 and A accumulated continuously during the cell cycle and were degraded at mitosis. Newborn cells had similar to 0.5% of the cyclin B1 content of dividing cells. Conclusion: The expression patterns of cyclins A, B1, and E can be explained by constant mRNA levels during the cell cycle. Previously reported phase specific variations of the cyclins are not strictly necessary for cell-cycle progression. Cells produced by membrane-elution are available to other laboratories for analysis of the cell cycle.