Flow cytometry study of human cyclin B1 and cyclin E expression in leukemic cell lines: Cell cycle kinetics and cell localization

Experiments by flow cytometry (FCM) after nuclei isolation have never been done to investigate cyclins. We have conducted different experiments by FCM using whole cells and isolated nuclei to study the immunolocalization and kinetic patterns of cyclin B1 and cyclin E in various leukemic cell lines. During asynchronous growth, all whole cells had a scheduled, cell cycle pha se-restricted expression of cyclin B1. By using a washless immunostaining o f unfixed nuclei, cyclin B1 was detected in all cell cycle phases, includin g G(1), although to a lesser extent than in G(2)/M, suggesting that in whol e cells the cyclin B1 epitope is masked and accessible only in isolated nuc lei. When the cells were synchronized at the G(1)/S boundary by thymidine o r in the G(1) phase by sodium n-butyrate, an identical accumulation of cycl in B1 was observed, As for cyclin E, its expression was higher with thymidi ne treatment than with sodium n-butyrate, particularly in nuclei. The eleva ted cyclin B1 level in the cells arrested at the G(1)/S boundary may reflec t the increased half-life of this protein stabilized as the result of cycli n E overexpression. However, our FCM data also support the notion that accu mulation of human cyclin B1 in leukemic cell lines begins during the G(1) p hase of the cell cycle, probably in the nucleus. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle confir ms this finding. It is possible, therefore, that tumor transformation or le ukemic phenotype may invariably be associated with altered cyclin B1 expres sion. (C) 1999 Academic Press.