Bcl-2 blocks 2-methoxyestradiol induced leukemia cell apoptosis by a p27(Kip1)-dependent G1/S cell cycle arrest in conjunction with NF-kappaB activation.
2-Methoxyestradiol (2-ME2) induces leukemia cells to undergo apoptosis in association with Bcl-2 inactivation but the mechanisms whereby Bcl-2 contributes to protection against programmed cell death in this context remain unclear. Here we showed that 2-ME2 inhibited the proliferation of Jurkat leukemia cells by markedly suppressing the levels of cyclins D3 and E, E2F1 and p21(Cip1/Waf1) and up-regulating p16(INK4A). Further, 2-ME2 induced apoptosis of Jurkat cells in association with down-regulation and phosphorylation of Bcl-2 (as mediated by JNK), up-regulation of Bak, activation of caspases-9 and -3 and PARP-1 cleavage. To determine the importance and mechanistic role of Bcl-2 in this process, we enforced its expression in Jurkat cells by retroviral transduction. Enforcing Bcl-2 expression in Jurkat cells abolished 2-ME2-induced apoptosis and instead produced a G1/S phase cell cycle arrest in association with markedly increased levels of p27(Kip1). Bcl-2 and p27(Kip1) were localized mainly in the nucleus in these apoptotic resistant cells. Interestingly, NF-kappaB activity and p50 levels were increased by 2-ME2 and suppression of NF-kappaB signaling reduced p27(Kip1) expression and sensitized cells to 2-ME2-induced apoptosis. Importantly, knocking-down p27(Kip1) in Jurkat Bcl-2 cells sensitized them to spontaneous and 2-ME2-induced apoptosis. Thus, Bcl-2 prevented the 2-ME2-induced apoptotic response by orchestrating a p27(Kip1)-dependent G1/S phase arrest in conjunction with activating NF-kappaB. Thus, we achieved a much better understanding of the penetrance and mechanistic complexity of Bcl-2 dependent anti-apoptotic pathways in cancer cells and why Bcl-2 inactivation is so critical for the efficacy of apoptosis and anti-proliferative inducing drugs like 2-ME2.
Batsi
Christina
C
Cell and Molecular Physiology Unit, Laboratory of Physiology, School of Medicine, University of Ioannina, 45110 Ioannina, Greece.
Markopoulou
Soultana
S
Kontargiris
Evangelos
E
Charalambous
Christiana
C
Thomas
Christoforos
C
Christoforidis
Savvas
S
Kanavaros
Panagiotis
P
Constantinou
Andreas I
AI
Marcu
Kenneth B
KB
Kolettas
Evangelos
E
D000077584:2-Methoxyestradiol; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D019169:Jurkat Cells / Q000166:cytology / Q000187:drug effects*; D016328:NF-kappa B / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D016196:S Phase / Q000187:drug effects / Q000502:physiology*
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004958:Estradiol; D000077584:2-Methoxyestradiol; D018844:Cyclin-Dependent Kinases