Regulation of p27Kip1 accumulation in murine B-lymphoma cells: role of c-Myc and calcium.

IgM cross-linking induces G1 arrest and apoptosis in murine B-lymphoma cells. It prevents pRb phosphorylation by decreasing cyclin-dependent kinase 2 activity via the up-regulation of cyclin kinase inhibitor p27Kip1. Anti-IgM also causes an increase in cytosolic free calcium and a loss of c-myc mRNA and protein. This down-regulation of c-Myc is prevented by CD40L, which rescues cells from anti-IgM-induced apoptosis. In this study, we addressed the mechanism(s) of anti-IgM-induced p27Kip1 accumulation. We examined effects of early events in B-cell receptor-mediated signaling, c-Myc down-regulation, and an increase in free calcium on p27Kip1. Down-regulation of c-myc alone had no effect on p27Kip1; neither did an increase in free calcium alone. Together, these two events led to p27Kip1 induction, growth arrest, and apoptosis. CD40L, the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, and cyclosporin A all prevented anti-IgM-induced p27Kip1 accumulation, suggesting that both the decrease in c-Myc expression and an increase in free calcium are necessary for p27Kip1 up-regulation.     

Environmental chemical-induced pro/pre-B cell apoptosis: analysis of c-Myc,p27Kip1, and p21WAF1 reveals a death pathway distinct from clonal deletion

Polycyclic aromatic hydrocarbons (PAH) are common environmental pollutants that suppress the immune system in part by inducing pro/pre-B cell apoptosis. The PAH-induced death signaling pathway resembles the signaling cascade activated during clonal deletion and modeled by B cell receptor cross-linking or by dexamethasone exposure of immature surface Ig(+) B cells in that apoptosis is mediated by NF-kappa B down-regulation. Because a PAH-induced, clonally nonrestricted deletion of B cells would have important implications for B cell repertoire development, the nature of the PAH-induced intracellular death signal was studied further. Particular emphasis was placed on the roles of growth arrest and c-Myc, p27(Kip1), and p21(WAF1) expression, because all of these elements contribute to clonal deletion. As in clonal deletion models, and as predicted by the down-regulation of NF-kappa B, PAH-induced death of pro/pre-B cells was at least partially dependent on c-Myc down-regulation. Furthermore, whereas dexamethasone induced a G(0)/G(1) cell cycle arrest, PAH had no effect on pro/pre-B cell growth, indicating that growth arrest and apoptosis occur by separable signaling pathways in this early phase of B cell development. Finally, in contrast to clonal deletion, PAH-induced pro/pre-B cell death was not dependent on p27(Kip1) or p21(WAF1) up-regulation but did coincide with p53 induction. These results distinguish the PAH-induced apoptosis pathway from that activated during clonal deletion and indicate that signaling cascades leading to growth arrest and/or apoptosis in pro/pre-B cells differ from those active at later B cell developmental stages.     

Increased p27Kip1 cyclin-dependent kinase inhibitor gene expression following anti-IgM treatment promotes apoptosis of WEHI 231 B cells

Engagement of the B cell receptor of WEHI 231 immature B cells leads sequentially to a drop in c-Myc, to induction of the cyclin-dependent kinase inhibitor p27Kip1, and finally to apoptosis. Recently we demonstrated that the drop in c-Myc expression promotes cell death, whereas the induction of p27 has been shown to lead to growth arrest. In this paper, we demonstrate that increased p27 expression also promotes apoptosis of WEHI 231 B cells. The rescue of WEHI 231 cells by CD40 ligand engagement of its receptor prevented the increase in p27 induction. Inhibition of p27-ablated apoptosis induced upon expression of antisense c-myc RNA. Furthermore, specific induction of p27 gene expression resulted in apoptosis of WEHI 231 cells. Lastly, inhibition of expression of c-Myc, upon induction of an antisense c-myc RNA vector, was sufficient to induce increased p27 levels and apoptosis. Thus, these findings define a signaling pathway during B cell receptor engagement in which the drop in c-Myc levels leads to an increase in p27 levels that promotes apoptosis.     

Role of phosphatidylinositol 3-kinase in anti-IgM- and anti-IgD-induced apoptosis in B cell lymphomas

Cross-linking of surface Ig receptors with anti IgM (anti-mu heavy chain, anti-mu), but not anti-IgD (anti-delta heavy chain, anti-delta), Abs leads to growth arrest and apoptosis in several extensively characterized B cell lymphomas. By poorly understood mechanisms, both Igs transiently stimulate c-Myc protein expression. However, ultimately, only anti-mu causes a severe loss in c-Myc and a large induction of p27(Kip1) protein expression. Because phosphatidylinositol 3-kinase (PI3K) has been established as a major modulator of cellular growth and survival, we investigated its role in mediating anti-Ig-stimulated outcomes. Herein, we show that PI3K pathways regulate cell cycle progression and apoptosis in the ECH408 B cell lymphoma. Anti-mu and anti-delta driven c-Myc protein changes precisely follow their effects on the PI3K effector, p70(S6K). Upstream of p70(S6K), signaling through both Ig receptors depresses PI3K pathway phospholipids below control with time, which is followed by p27(Kip1) induction. Conversely, anti-delta, but not anti-mu stimulated PI3K-dependent phospholipid return to control levels by 4-8 h. Abrogation of the PI3K pathway with specific inhibitors mimics anti-mu action, potentiates anti-mu-induced cell death and, importantly, converts anti-delta to a death signal. Transfection with active PI3K kinase construct induces anti-mu resistance, whereas transfection with dominant negative PI3K augments anti-mu sensitivity. Our results show that prolonged disengagement of PI3K or down-regulation of its products by anti-mu (and not anti-delta) determines B cell fate.     

Pancreatic small cells: analysis of quiescence, long-term maintenance and insulin expression in vitro

We have previously identified a novel population of small cells in human and canine pancreas characterized by immature morphology, quiescence, and a glucose-responsive insulin secretion. Based on their immature phenotype and predominant presence in small islets, we have hypothesized that small cells serve as islet progenitors. This hypothesis remains untested, however, due to persistent quiescence and scarcity of small cells in vitro. We have recently developed a culture medium that allowed for modest small cell proliferation. In this study we characterized the expression of genes potentially involved in small cell growth regulation by Q-RT-PCR. Our results suggest that quiescence of small cells correlates with up-regulation of Cdk inhibitors p27(Kip1), p16(INK4a) and p21(CIP1), PTEN, Hep27 and Foxo1a and with down-regulation of c-Myc and the receptors for EGF, FGF2 and HGF. The exit from quiescence correlates with activation of EGFR expression and down-regulation of p27(Kip1) and p16(INK4a). We also report here that small cells can be maintained in long-term non-adherent cultures preserving insulin and glucagon production for up to 208 days. Therefore, expansion of small cells in vitro may have a significant potential for the treatment of diabetes. This study is an important step in understanding the mechanisms involved in small cell growth regulation, which is required to fully evaluate their functional potential.     

CD40 ligand rescues inhibitor of differentiation 3-mediated G1 arrest induced by anti-IgM in WEHI-231 B lymphoma cells

The engagement of membrane-bound Igs (mIgs) results in growth arrest, accompanied by apoptosis, in the WEHI-231 murine B lymphoma cells, a cell line model representative of primary immature B cells. Inhibitor of differentiation (Id) proteins, members of the helix-loop-helix protein family, functions in proliferation, differentiation, and apoptosis in a variety of cell types. In this study, we analyzed the involvement of Id protein in mIg-induced growth arrest and apoptosis in WEHI-231 cells. Following stimulation with anti-IgM, expression of Id3 was up-regulated at both the mRNA and protein levels; this up-regulation could be reversed by CD40L treatment. Retrovirus-mediated transduction of the Id3 gene into WEHI-231 cells resulted in an accumulation of the cells in G(1) phase, but did not induce apoptosis. E box-binding activity decreased in response to anti-IgM administration, but increased after stimulation with either CD40L alone or anti-IgM plus CD40L, suggesting that E box-binding activity correlates with cell cycle progression. WEHI-231 cells overexpressing Id3 accumulated in G(1) phase, which was accompanied by reduced levels of cyclin D2, cyclin E, and cyclin A, and a reciprocal up-regulation of p27(Kip1). Both the helix-loop-helix and the C-terminal regions of Id3 were required for growth-suppressive activity. These data suggest that Id3 mimics mIg-mediated G(1) arrest in WEHI-231 cells.     

Differential c-Myc responsiveness to B cell receptor ligation in B cell-negative selection

Responsiveness of c-Myc oncogene to B cell receptor ligation has been implicated in the induction of apoptosis in transformed and normal immature B cells. These studies provided compelling evidence to link the c-Myc oncogene with the process of negative selection in B-lymphocytes. However, in addition to apoptosis, B cell-negative selection has been shown to occur by secondary Ig gene rearrangements, a mechanism called receptor editing. In this study, we assessed whether differential c-Myc responsiveness to B cell receptor (BCR) ligation is associated with the mechanism of negative selection in immature B cells. Using an in vitro bone marrow culture system and an Ig-transgenic mouse model (3-83) we show here that c-Myc is expressed at low levels throughout B cell development and that c-Myc responsiveness to BCR ligation is developmentally regulated and increased with maturation. Furthermore, we found that the competence to mount c-Myc responsiveness upon BCR ligation is important for the induction of apoptosis and had no effect on the process of receptor editing. Therefore, this study suggests an important role of c-Myc in promoting and/or maintaining B cell development and that compartmentalization of B cell tolerance may also be developmentally regulated by differential c-Myc responsiveness.     

RasGRP1 sensitizes an immature B cell line to antigen receptor-induced apoptosis

RasGRP1 is a guanine nucleotide exchange factor that activates Ras GTPases and is activated downstream of antigen receptors on both T and B lymphocytes. Ras-GRP1 provides signals to immature T cells that confer survival and proliferation, but RasGRP1 also promotes T cell receptor-mediated deletion of mature T cells. We used the WEHI-231 cell line as an experimental system to determine whether RasGRP1 can serve as a quantitative modifier of B cell receptor-induced deletion of immature B cells. A 2-fold elevation in RasGRP1 expression markedly increased apoptosis of WEHI-231 cells following B cell receptor ligation, whereas a dominant negative mutant of RasGRP1 suppressed B cell receptor-induced apoptosis. Activation of ERK1 or ERK2 kinases was not required for RasGRP1-mediated apoptosis. Instead, elevated RasGRP1 expression caused down-regulation of NF-kappaB and Bcl-x(L), which provide survival signals counter-acting apoptosis induction by B cell receptor. Inhibition of NF-kappaB was sufficient to enhance B cell receptor-induced apoptosis of WEHI-231 cells, and ligation of co-stimulatory receptors that activate NF-kappaB suppressed the ability of RasGRP1 to promote B cell receptor-induced apoptosis. These experiments define a novel apoptosis-promoting pathway leading from B cell receptor to the inhibition of NF-kappaB and demonstrate that differential expression of RasGRP1 has the potential to modulate the sensitivities of B cells to negative selection following antigen encounter.     

CD27 and CD40 inhibit p53-independent mitochondrial pathways in apoptosis of B cells induced by B cell receptor ligation

B cells in the germinal center are known to undergo apoptosis after B cell receptor (BCR) ligation, a process relevant to immunological tolerance. Human CD27 is a B cell co-stimulatory molecule. The aim of this study was to compare the effects of CD27 and CD40 signals on BCR-mediated apoptosis of B cells. BCR ligation activated mitochondrial apoptotic pathways including down-regulation of Bcl-X(L), dissipation of mitochondrial transmembrane potential, release of cytochrome c, and activation of caspase-9. Each of these effects was significantly inhibited by CD27 and CD40. Bik expression was weakly but significantly down-regulated by CD27 but up-regulated by CD40. BCR ligation resulted in p53 activation including its phosphorylation at Ser(15), nuclear translocation, and target gene p53AIP1 induction. CD27 and CD40 clearly suppressed these processes. Analyses that used dominant-negative p53 variants revealed a low but still substantial level of BCR-mediated apoptosis and intact mitochondria-mediated apoptotic pathway. These pathways were further inhibited by CD27 and CD40, although the cells showed no p53 phosphorylation or p53AIP1 expression. Our results suggested that, at the mitochondrial level, CD27 and CD40 co-stimulatory signals regulated the p53-amplified apoptotic pathway in B cells through the inhibition of p53-independent apoptotic pathway primarily induced by BCR ligation.     

CD72 down-modulates BCR-induced signal transduction and diminishes survival in primary mature B lymphocytes

CD72, a 45-kDa type II transmembrane glycoprotein carrying an ITIM motif, is believed to be an inhibitory coreceptor of the BCR. Mature B cells lacking CD72 show enhanced Ca(2+) mobilization and are hyperproliferative in response to BCR ligation. However, the signal transduction pathways downstream of BCR signaling that transmit the inhibitory effect of CD72 in mature B cells remain unknown. To address this question, we used hen egg lysozyme-specific BCR transgenic mice to elucidate the differential cell signaling between wild-type and CD72-deficient B cells in response to hen egg lysozyme Ag stimulation. Our results demonstrate that CD72 predominantly down-regulates the major signal transduction pathways downstream of the BCR, including NF-AT, NF-kappaB, ERK, JNK, p38-MAPK, and PI3K/Akt in mature B cells. CD72 ligation with anti-CD72 Ab (K10.6), which mimics the binding of CD100 (a natural ligand for CD72) to release the inhibitory function of CD72, augments cell proliferation, Ca(2+) flux, IkappaBalpha activation, and ERK MAPK activity upon Ag stimulation in wild-type B cells. In addition, we show direct evidence that CD72 promotes cell cycle arrest and apoptosis after Ag stimulation in mature B cells. Taken together, our findings conclude that CD72 plays a dominant role as a negative regulator of BCR signaling in primary mature B lymphocytes.     

c-Myc sensitizes cells to tumor necrosis factor-mediated apoptosis by inhibiting nuclear factor kappa B transactivation

Nuclear factor kappaB (NF-kappaB) plays a key role in suppression of tumor necrosis factor (TNF)-mediated apoptosis by inducing a variety of anti-apoptotic genes. Expression of c-Myc has been shown to sensitize cells to TNF-mediated apoptosis by inhibiting NF-kappaB activation. However, the precise step in the NF-kappaB signaling pathway and apoptosis modified by c-Myc has not been identified. Using the inducible c-MycER system and c-Myc null fibroblasts, we found that expression of c-Myc inhibited NF-kappaB activation by interfering with RelA/p65 transactivation but not nuclear translocation of NF-kappaB. Activation of c-Myc promoted TNF-induced release of cytochrome c from mitochondria to the cytosol because of the inhibition of NF-kappaB. Furthermore, we found that NF-kappaB-inducible gene A1 was attenuated by expression of c-Myc and that the restoration of A1 expression suppressed c-Myc-induced TNF sensitization. Our results elucidate the molecular mechanisms by which c-Myc increases cell susceptibility to TNF-mediated apoptosis, indicating that c-Myc may exhibit its pro-apoptotic activities by repression of cell survival genes.     

Involvement of cell cycle progression in survival signaling through CD40 in the B-lymphocyte line WEHI-231

The CD40 molecule transmits a signal that abrogates apoptosis induced by ligation of the antigen receptor (BCR) in both primary B cells and B-cell lines such as WEHI-231. Expression of Bcl-xL and A1, antiapoptotic members of the Bcl-2 family, is enhanced by CD40 ligation, and is suggested to mediate CD40-induced B-cell survival. CD40 ligation also promotes cell cycle progression by increasing the levels of cyclin-dependent kinases (CDKs) required for cell cycle progression, and reducing expression of the CDK inhibitor p27(kip1). Here we demonstrate that cell cycle inhibition by retrovirus-mediated p27(kip1) expression does not modulate the levels of Bcl-xL or A1, but significantly reduces the survival of BCR-ligated WEHI-231 cells by CD40 ligation. This indicates that cell cycle progression is crucial for CD40-mediated survival of B cells.     

Blockade of the extracellular signal-regulated kinase pathway induces marked G1 cell cycle arrest and apoptosis in tumor cells in which the pathway is constitutively activated: up-regulation of p27(Kip1)

Constitutive activation of the ERK pathway is associated with the neoplastic phenotype of a relatively large number of human tumor cells. Blockade of the ERK pathway by treatment with PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase/ERK kinase (MEK), completely suppressed the growth of tumor cells in which the pathway is constitutively activated (RPMI-SE and HT1080 cells). Consistent with its prominent antiproliferative effect, PD98059 induced a remarkable G(1) cell cycle arrest, followed by a modest apoptotic response, in these tumor cells. Selective up-regulation of p27(Kip1) was observed after PD98059 treatment of RPMI-SE and HT1080 cells. Overexpression in RPMI-SE cells of either a kinase-negative form of MEK1 or wild-type MAP kinase phosphatase-3 also induced up-regulation of p27(Kip1). The up-regulation of p27(Kip1) correlated with increased association of p27(Kip1) with cyclin E-cyclin-dependent kinase (CDK) 2 complexes, a concomitant inhibition of cyclin E-CDK2 kinase activity, and a consequent decrease in the phosphorylation state of retinoblastoma protein, which would culminate in the marked G(1) cell cycle arrest observed in these tumor cells. These results suggest that the complete growth suppression that follows specific blockade of the ERK pathway in tumor cells in which the pathway is constitutively activated is mediated by up-regulation of p27(Kip1).