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) m ethods that accumulation of human cyclin B1 in leukemic cell lines begins d uring the G(1) phase of the cell cycle (Viallard ct al., Exp Cell Res 247: 208-219, 1999). In the present study, FCM was used to compare the localizat ion and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cel l 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. Ce lls (leukemic cell line Jurkat or PKA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was use d to confirm certain results. Results: Under asynchronous growing conditions and for both cell population s, cyclin B1 expression was essentially restricted to the G(2)/M transition , reaching its maximal level at mitosis. When the cells were synchronized a t the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-but yrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lym phocytes ex-pressed cyclin B1 only during the thymidine block. The cyclin B 1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly simila r when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were c onfirmed 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 lymphocy tes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defe ctive cyclin B1 proteolysis. Conclusions: We found that the leukemic T cells behaved quite differently f rom 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 cel ls but not in normal T lymphocytes. Therefore, the restriction point from w hich cyclin B1 can be detected is different in the two models studied. We h ypothesize that after passage through a restriction point differing in T ly mphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes co nstant in the S and G(2)/M phases and independent from the DNA replication cycle. Cytometry 39:117-125, 2000. (C) 2000 Wiley-Liss, Inc.