Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells.
BACKGROUND
In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes.


METHODS
Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results.


RESULTS
Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis.


CONCLUSIONS
We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.
Viallard
J F
JF
Laboratoire de Greffe de Moelle, UMR-CNRS 5540, Bordeaux, France.
Lacombe
F
F
Dupouy
M
M
Ferry
H
H
Belloc
F
F
Reiffers
J
J
D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology; D002455:Cell Division; D003078:Colchicine / Q000494:pharmacology; D019926:Cyclin B / Q000378:metabolism*; D056744:Cyclin B1; D004247:DNA / Q000032:analysis; D004261:DNA Replication; D005434:Flow Cytometry; D006801:Humans; D007399:Interphase; D019169:Jurkat Cells / Q000378:metabolism; D008213:Lymphocyte Activation; D008938:Mitosis; D010835:Phytohemagglutinins / Q000494:pharmacology; D016196:S Phase; D013601:T-Lymphocytes / Q000378:metabolism*; D013936:Thymidine / Q000494:pharmacology
D002087:Butyrates; C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D010835:Phytohemagglutinins; D004247:DNA; D003078:Colchicine; D013936:Thymidine