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 phase-restricted expression of cyclin B1. By using a washless immunostaining of unfixed nuclei, cyclin B1 was detected in all cell cycle phases, including G1, although to a lesser extent than in G2/M, suggesting that in whole cells the cyclin B1 epitope is masked and accessible only in isolated nuclei. When the cells were synchronized at the G1/S boundary by thymidine or in the G1 phase by sodium n-butyrate, an identical accumulation of cyclin B1 was observed. As for cyclin E, its expression was higher with thymidine treatment than with sodium n-butyrate, particularly in nuclei. The elevated cyclin B1 level in the cells arrested at the G1/S boundary may reflect the increased half-life of this protein stabilized as the result of cyclin E overexpression. However, our FCM data also support the notion that accumulation of human cyclin B1 in leukemic cell lines begins during the G1 phase of the cell cycle, probably in the nucleus. The detection of cyclin B1 by Western blot in cells sorted in the G1 phase of the cell cycle confirms this finding. It is possible, therefore, that tumor transformation or leukemic phenotype may invariably be associated with altered cyclin B1 expression.
Laboratoire de Greffe de Moelle, UMR-CNRS 5540, 146 rue Léo Saignat, Bordeaux, 33076, France.
J F
JF
Viallard
F
F
Lacombe
M
M
Dupouy
H
H
Ferry
F
F
Belloc
J
J
Reiffers
D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division; D002469:Cell Separation; D019926:Cyclin B / Q000096:biosynthesis* / Q000378:metabolism; D056744:Cyclin B1; D019927:Cyclin E / Q000096:biosynthesis* / Q000378:metabolism; D005434:Flow Cytometry; D016193:G1 Phase; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D007700:Kinetics; D007938:Leukemia / Q000378:metabolism* / Q000473:pathology*; D014407:Tumor Cells, Cultured; D020298:U937 Cells
C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D019927:Cyclin E