Prevention of Anti-IgM-Induced Apoptosis Accompanying G1 Arrest in B Lymphoma Cells Overexpressing Dominant-Negative Mutant Form of c-Jun N-Terminal Kinase 1

Abstract

A family of mitogen-activated protein (MAP) kinases comprising the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 MAP kinases are involved in proliferation and apoptosis. However, there are some arguments concerning the role of these kinases in Ag-induced B cell apoptosis. Two of the B lymphoma cell lines (CH31 and WEHI-231) susceptible to anti-IgM-induced apoptosis were used as a model. To address these issues, we examined the kinetics of anti-IgM-induced activation of MAP kinases and established cell lines overexpressing a dominant-negative (dn) mutant form of JNK1 (dnJNK1). Anti-IgM induced a sustained JNK1 activation with a peak at 8 h, with a marginal activation of ERK1/ERK2 in CH31 cells. The sustained JNK1 activation was not a secondary event through a caspase activation. The peak point of the JNK1 activation was just before the onset of a decline in mitochondrial membrane potential, which preceded anti-IgM-induced cell death. Following anti-IgM stimulation, dnJNK1 prevented a decline in mitochondrial membrane potential at 24 h, with a prolonged inhibition up to 72 h in WEHI-231, although it did so only partially during a later time period in CH31. The dnJNK1 cells also demonstrated diminished procaspase-3 activation and a decreased rate of apoptosis upon anti-IgM stimulation, with a concomitant increased arrest in G1 phase, which could be explained by enhanced levels of cyclin-dependent kinase inhibitor p27Kip1 protein. Thus, anti-IgM-induced JNK activation might be implicated in cell cycle progression as well as in apoptosis regulation, probably involving p27Kip1 protein.

Chronic Ethanol Exposure Potentiates Lipopolysaccharide Liver Injury Despite Inhibiting Jun N-terminal Kinase and Caspase 3 Activation

“…Indeed, in this in vivo model of alcohol-related liver damage, there is compelling evidence that prior ethanol exposure inhibits classical apoptotic signaling because it prevents the activation of both procaspase 8 and JNK. The former normally functions as an initiator caspase for death receptor-induced apoptosis, and the latter is necessary for stress-induced procaspase 3 activation and subsequent apoptosis in many cell types (32)(33)(34)(35)(36)66). Consistent with this concept, we were unable to demonstrate increased cytochrome c release or nuclear oligonucleosomal DNA fragmentation in LPS-treated ethanol-fed mice, although these animals developed worse liver damage than the PF controls.…”

“…Recent reports suggest that Jun N-terminal kinase (JNK), the terminal enzyme in the TNF-regulated, stress-activated kinase cascade, is required for procaspase 3 activation (32,33). This finding complements other evidence demonstrating that JNK activation is necessary for apoptosis in many cells types (34 -36).…”

Tumor necrosis factor-related apoptosis-inducing ligand induces apoptotic cell death through c-Jun NH2-terminal kinase activation in squamous cell carcinoma cells

“…At each passage, cells were harvested as single cell suspensions using trypsin/EDTA. Several stable cell lines expressing the dominant-negative form of JNK1 (dnJNK1) were established by a previously described procedure (21). Briefly, MIT6 cells were transfected with an expression vector containing dnJNK1 or a control vector alone, and cultured in the presence of G418.…”

“…Cells were transfected with expression vectors encoding dnJNK1 or control vector alone (21), then cloned by limiting dilution to obtain clone(s). Expression of dnJNK1 was confirmed by Western blotting.…”

“…To explore the requirement for JNK1 activation in the TRAIL-induced cytotoxicity in SCC cells, a line of TRAIL-sensitive MIT6 cells overexpressing dnJNK1 was established using the procedure as previously described (20,21). One (DN12) of several clones was demonstrated to express exogenous dnJNK1 (p47) as well as a major endogenous form of JNK1 (p46) (Fig.…”