p53 mediates apoptosis induced by c-Myc activation in hypoxic or gamma irradiated fibroblasts

Deregulated c-Myc expression leads to a cellular state where proliferation and apoptosis are equally favored depending on the cellular microenvironment. Since the apoptotic sensitivity of many cells is influenced by the status of the p53 tumor suppressor gene, we investigated whether the induction of apoptosis by DNA damage or non-genotoxic stress are also influenced by the p53 status of cells with altered c-Myc activity. Rat-1 fibroblasts expressing a conditional c-Myc allele (c-MycER), were transfected to express an antisense RNA complimentary to p53 mRNA. Expression of antisense p53 RNA decreased p53 protein levels and delayed p53 accumulation following c-Myc activation. Under hypoxic or low serum conditions, cells expressing antisense p53 were substantially more resistant to c-Myc-induced apoptosis than were control cells. c-Myc activation also sensitized Rat-1 cells to radiation-induced apoptosis. Rat-1 cells expressing antisense p53 RNA were more resistant to apoptosis induced by the combined effects of c-Myc activation and gamma irradiation. In a similar manner, apoptosis induced by c-Myc in serum starved, hypoxic or gamma irradiated fibroblasts was also inhibited by Bcl-2. These data indicate that p53 is involved in c-Myc-mediated apoptosis under a variety of stresses which may influence tumor growth, evolution and response to therapy.     

CD72 stimulation modulates anti-IgM induced apoptotic signaling through the pathway of NF-kappaB, c-Myc and p27(Kip1)

Engagement of mIgM induces G1 arrest and apoptosis in immature B cells. The biochemical mechanism(s) regulating the cell death process are poorly understood. Cross-linking of CD72 (a B cell co-receptor) with anti-CD72 antibody was shown to protect B cells from apoptosis. We investigated the molecular mechanism involved in apoptosis preventing signaling mediated by CD72 ligation using a derivative (WEHIdelta) of the WEHI231 cell line which is representative of immature B cells. Apoptotic WEHIdelta cells following cross-linking of mIgM demonstrate a dramatic loss of c-Myc protein after transient up-regulation. In contrast, pre-ligation of CD72 was able to sustain c-Myc expression after transient up-regulation. Cross-linking of mIgM of WEHIdelta cells causes accumulation of the Cdk inhibitor, p27(Kip1). CD72 pre-ligation was shown to inhibit the accumulation of p27(Kip1) protein. Moreover, NF-kappaB activity was not suppressed in WEHIdelta cells after mIgM cross-linking when the cells were pre-treated with anti-CD72 antibody. These results strongly suggest that the apoptosis preventing signal evoked by CD72 ligation is delivered through the pathway of NF-kappaB, c-Myc, p27(Kip1) and cyclin.     

Inhibition of NF-kappaB/Rel induces apoptosis of murine B cells.

Apoptosis of the WEHI 231 immature B cell lymphoma line following membrane interaction with an antibody against the surface IgM chains (anti-IgM) is preceded by dramatic changes in Nuclear Factor-kappaB (NF-kappaB)/ Rel binding activities. An early transient increase in NF-kappaB/Rel binding is followed by a significant decrease in intensity below basal levels. Here we have explored the role of these changes in Rel-related factors in B cell apoptosis. Treatment of WEH1 231 cells with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a protease inhibitor which prevents degradation of the inhibitor of NF-kappaB (IkappaB)-alpha, or with low doses of pyrrolidinedithiocarbamate (PDTC) selectively inhibited NF-kappaB/Rel factor binding and induced apoptosis. Bcl-XL expression protected WEHI 231 cells from apoptosis induced by these agents. Microinjection of WEHI 231 cells with either IkappaB-alpha-GST protein or a c-Rel affinity-purified antibody induced apoptosis. Ectopic c-Rel expression ablated apoptosis induced by TPCK or anti-IgM. Treatment of BALENLM 17 and A20 B lymphoma cells or normal murine splenic B lymphocytes with either TPCK or PDTC also resulted in apoptosis. These findings indicate that the drop in NF-kappaB/Rel binding following anti-IgM treatment activates apoptosis of WEHI 231 cells; furthermore, they implicate the NF-kappaB/Rel family in control of apoptosis of normal and transformed B cells.     

Reduction of c-myc expression by an antisense approach under Cre/loxP switching induces apoptosis in human liver cancer cells

c-Myc has been documented to be both a positive and a negative signal for the induction of apoptosis. It is well known that overexpression of the c-myc gene induces apoptosis of normal cells, but the result of a reduction in its expression is not fully understood. We examined whether a reduction in c-myc expression would induce apoptosis in human liver cancer cells. Specifically, antisense and sense oligodeoxynucleotides (oligos) against the human c-myc mRNA were synthesized, mixed with a liposome reagent at various ratios, and were applied to the liver cancer-derived cell lines, HCC-T, HepG2, and PLC/PRF/5. To exclude effects resulting from using oligos, plasmid vectors expressing the full-length c-myc cDNA in both sense and antisense orientations under the control of the Cre/loxP system were generated. Monoclonal cell lines including these plasmid vectors were produced and Cre was supplied by adenovirus infection. Apoptosis was determined morphologically and c-Myc and Bcl-2 expression was examined by Western blotting. The antisense myc significantly inhibited the proliferation of the cells within two days, while neither the liposome reagent alone nor sense myc did so. Most of the cells were rounded up by the antisense-treatment and nuclear fragmentation and DNA ladder formation were detected after two days in antisense c-myc-treated cells. Antisense c-myc largely reduced c-Myc and partially Bcl-2 expression; overexpression of Bcl-2 partially rescued from apoptosis in HCC-T and HepG2 cells. These results suggest that the massive reduction in c-myc mRNA induces apoptosis in liver cancer cell lines and consequent decrease in Bcl-2 enhances the cell death. c-Myc reduction under the Cre/loxP switching system may be a useful tool for the clarification of c-myc-related cellular mechanisms in differentiation and proliferation.     

Prostaglandin EP4 receptor enhances BCR-induced apoptosis of immature B cells

Prostaglandin E2 (PGE2) is emerging as an important co-modulator of B cell responses. Using a pharmacological approach, we aimed to delineate the role of PGE2 in B cell receptor (BCR) induced apoptosis of immature B cells. Gene and protein expression analyses showed that, of the four PGE2 receptors subtypes, only EP4 receptor is upregulated upon BCR cross-linking, leading to sensitization of WEHI 231 cells towards PGE2 mediated inhibitory effects. EP4 receptor antagonist ONO-AE3-208, was able to completely revert the observed effects of PGE2. The engagement of EP4 receptor promotes BCR-induced G0/G1 arrest of WEHI 231 cells, resulting in enhanced caspase mediated, BCR-induced apoptosis. We addressed, mechanistically, the interplay between BCR and EP4 receptor signaling components. Prostaglandin1-alcohol (Pge1-OH), a selective EP4 receptor agonist inhibits BCR-induced activation of NF-κB by suppression of BCR-induced IκBα phosphorylation. Disruption of prosurvival pathways is a possible mechanism by which PGE2 enhances BCR-induced apoptosis in immature B lymphocytes.     

The novel adaptor protein Swiprosin-1 enhances BCR signals and contributes to BCR-induced apoptosis

B-cell receptor (BCR) signals are essential for B-cell differentiation, homeostasis and negative selection, which are regulated by the strength and quality of BCR signals. Recently, we identified a new adaptor protein, Swiprosin-1, in lipid rafts of B-cell lines that undergo apoptosis after BCR stimulation. During murine B-cell development, Swiprosin-1 exhibited highest expression in immature B cells of the bone marrow, but was also expressed in resting and activated splenic B cells and in non-lymphoid tissue, especially in the brain. Ectopic expression of Swiprosin-1 in the immature murine B-cell line WEHI231 enhanced spontaneous and BCR-induced apoptosis. In contrast, short hairpin RNA (shRNA)-mediated downregulation of Swiprosin-1 impaired specifically spontaneous and BCR-elicited apoptosis, but not BCR-induced G1 cell cycle arrest and upregulation of the cell cycle inhibitor p27(Kip1). In accordance, Swiprosin-1 abundance regulated net cell growth of WEHI231 cell populations through reciprocal regulation of Bcl-xL, but not Bim, thereby controlling spontaneous apoptosis. Swiprosin-1-enhanced apoptosis was blocked through nuclear factor kappaB-activating stimuli, namely B-cell-activating factor of the TNF family, anti-CD40 and lipopolysaccharide (LPS). This correlated with enhanced BCR-induced IkappaB-alpha phosphorylation and degradation in cells expressing a Swiprosin-1-specific shRNA. Finally, ectopic Swiprosin-1 expression enhanced BCR-induced cell death in primary, LPS-stimulated splenic B cells. Hence, Swiprosin-1 may regulate lifespan and BCR signaling thresholds in immature B cells.     

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.     

p27 modulates cell cycle progression and chemosensitivity in human malignant glioma.

The cell cycle regulatory protein p27, an inhibitor of cyclin-dependent kinases (CDK), has been attributed a role in (i) prognosis in breast and colon cancer, (ii) induction of apoptosis in cancer cells, and (iii) resistance to cancer chemotherapy. Here we report that p27 is widely expressed in human malignant gliomas in vivo and in glioma cell lines in vitro. Serum deprivation or confluency promotes p27 protein accumulation in vitro. Neither baseline p27 levels nor p27 levels induced by confluency or serum deprivation correlate with p53 status or drug sensitivity of human glioma cell lines. Expression of antisense p27 mRNA increased the doubling times in T98G glioma cells, whereas sense p27 mRNA had no such effect. There was a density-dependent and drug-specific modulation of chemosensitivity by sense or antisense mRNA expression in T98G cells. Taken together, these data define a strong p27 response to altered growth conditions and suggest a role for p27 in modulating response to chemotherapy in human malignant glioma cells.     

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.