A proapoptotic signaling pathway involving RasGRP, Erk, and Bim in B cells.
OBJECTIVE
Bryostatin-1 and related diacylglycerol (DAG) analogues activate RasGRPs in lymphocytes, thereby activating Ras and mimicking some aspects of immune receptor signaling. To define the role of RasGRPs in lymphocyte apoptosis and to identify potential therapeutic uses for DAG analogues in lymphocyte disorders, we characterized the response of lymphoma-derived cell lines to DAG analogues.


METHODS
Human lymphoma-derived B cell lines and mouse primary B cells were treated with bryostatin-1 or its synthetic analogue "pico." Ras signaling partners and Bcl-2 family members were studied with biochemical assays. Cellular responses were monitored using growth and apoptosis assays.


RESULTS
Stimulation of B cells with DAG analogues results in activation of protein kinase C/RasGRP-Ras-Raf-Mek-Erk signaling and phosphorylation of the proapoptotic BH3-only protein Bim. In vitro, Bim is phosphorylated by Erk on sites previously associated with increased apoptotic activity. In Toledo B cells derived from a non-Hodgkin's lymphoma (B-NHL), DAG analogue stimulation leads to extensive apoptosis. Apoptosis can be suppressed by either downregulation of Bim or overexpression of Bcl-2. It is associated with the formation of Bak-Bax complexes and increased mitochondrial membrane permeability. Toledo B-NHL cell apoptosis shows a striking dependence on sustained signaling.


CONCLUSIONS
In B cells, Erk activation leads directly to phosphorylation of Bim on sites associated with activation of Bim. In Toledo B-NHL cells, the dependence of apoptosis on sustained signaling suggests that Bcl-2 family members could interpret signal duration, an important determinant of B cell receptor-mediated negative selection. Certain cases of B-NHL might respond to DAG analogue treatment by the mechanism outlined here.
Stang
Stacey L
SL
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Lopez-Campistrous
Ana
A
Song
Xiaohua
X
Dower
Nancy A
NA
Blumberg
Peter M
PM
Wender
Paul A
PA
Stone
James C
JC
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D001402:B-Lymphocytes / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D054713:Bryostatins / Q000494:pharmacology; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D051336:Mitochondrial Membranes / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C000606775:BCL2L11 protein, human; C494827:Bax protein, mouse; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; D054713:Bryostatins; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; C491790:RASGRP1 protein, human; C112278:Rasgrp1 protein, mouse; D011947:Receptors, Antigen, B-Cell; D051028:bcl-2-Associated X Protein; C046785:bryostatin 1; D048049:Extracellular Signal-Regulated MAP Kinases