Cycling B-CLL cells are highly susceptible to inhibition of the proteasome: involvement of p27, early D-type cyclins, Bax, and caspase-dependent and -independent pathways.
OBJECTIVE
Although peripheral blood B-CLL cells are arrested in G0 phase of the cell cycle, a proliferating pool of cells in proliferation centers might be involved in disease progression. We have previously described an in vitro model of this proliferating pool of cells using B-CLL cells stimulated with immunostimulatory oligonucleotides (CpG-ODN) and interleukin-2. Lactacystin is a specific inhibitor of the proteasome and is a potent apoptosis inductor in resting peripheral B-CLL cells. In the present study, we investigated the effect of proteasome inhibition in proliferating B-CLL cells.


METHODS
The effect of proteasome inhibition was analyzed using thymidine incorporation, annexin V assays, and TUNEL staining. Immunoblots were performed to evaluate expression of proteins involved in cell cycle and apoptosis regulation.


RESULTS
Lactacystin blocked cell cycle progression in activated B-CLL cells and inhibited degradation of p27. Upregulation of cyclin D2 and D3 in activated B-CLL cells was inhibited while the expression of cdk2, cdk4, and cyclin E remained unchanged. Activated B-CLL cells were more susceptible to apoptosis induction as compared to resting B-CLL cells. Apoptosis induction was accompanied by cleavage of Bax, procaspase 8, procaspase 9, and procaspase 3. However, a broad-spectrum caspase inhibitor (z-VAD.fmk) only partially inhibited cell death although DNA degradation was completely inhibited.


CONCLUSIONS
Proteasome inhibition is highly effective in proliferating B-CLL cells and induces apoptosis using a caspase-dependent and -independent pathway.
Bogner
Christian
C
IIIrd Department of Medicine, Technical University of Munich, Munich, Germany.
Schneller
Folker
F
Hipp
Susanne
S
Ringshausen
Ingo
I
Peschel
Christian
C
Decker
Thomas
T
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000187:drug effects; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000473:pathology*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D046988:Proteasome Endopeptidase Complex; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D025521:Tumor Suppressor Proteins / Q000187:drug effects; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D018797:Cell Cycle Proteins; D016213:Cyclins; D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D025521:Tumor Suppressor Proteins; D051028:bcl-2-Associated X Protein; C067713:lactacystin; D050760:Cyclin-Dependent Kinase Inhibitor p27; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine