c-Myc: where death and division collide

c-Myc is intimately involved in cell proliferation. However, inappropriate activation of c-Myc may also promote or sensitize cells to apoptosis. This is well established for several cell types and has been reinforced by a recent report in Cell Death and Differentiation by de Alboran and colleagues, who report that c-Myc-deficient B cells are resistant to several apoptotic stimuli, supporting a role for c-Myc in modulating B lymphocyte cell death. Here, we integrate these findings into the current picture of how c-Myc participates in cell death control.     

c-Myc: Where Death and Division Collide

c-Myc is intimately involved in cell proliferation. However, inappropriate activation of c-Myc may also promote or sensitize cells to apoptosis. This is well established for several cell types and has been reinforced by a recent report in Cell Death and Differentiation by de Alboran and colleagues, who report that c-Myc-deficient B cells are resistant to several apoptotic stimuli, supporting a role for c-Myc in modulating B lymphocyte cell death. Here, we integrate these findings into the current picture of how c-Myc participates in cell death control.     

Cutting edge: a novel mechanism for rescue of B cells from CD95/Fas-mediated apoptosis.

CD95-induced apoptosis contributes to the maintenance of homeostasis in both B and T lymphocyte-mediated immunity. B cells increase CD95 expression in response to activation signals and become susceptible to CD95-induced apoptosis. Protection from CD95-mediated death signals can be induced in mature B cells by signals delivered through the B cell Ag receptor. In this paper we demonstrate for the first time that rescue from apoptosis can occur independently of de novo protein synthesis. This rescue from apoptosis prevents activation of caspase 8, the apical caspase in the CD95 death pathway, and CD95-FADD (Fas-associated death domain containing protein) association does not occur normally. Thus B cell activation signals can biochemically modify proximal elements of the CD95 death pathway and regulate the sensitivity of cells to apoptosis induction at an early stage in programmed cell death.     

Signaling through MHC class II molecules blocks CD95-induced apoptosis

B cells are induced to express CD95 upon interaction with T cells. This interaction renders the B cells sensitive to CD95-mediated apoptosis, but ligation of proviability surface receptors is able to inhibit apoptosis induction. MHC class II is a key molecule required for Ag presentation to Th cells, productive T cell-B cell interaction, and B cell activation. We demonstrate here for the first time that MHC class II ligation also confers a rapid resistance to CD95-induced apoptosis, an affect that does not require de novo protein synthesis. Signaling through class II molecules blocks the activation of caspase 8, but does not affect the association of CD95 and Fas-associated death domain-containing protein. MHC class II ligation thus blocks proximal signaling events in the CD95-mediated apoptotic pathway.     

A role for c-myc in chemically induced renal-cell death.

A variety of genes, including c-myc, are activated by chemical toxicants in vivo and in vitro. Although enforced c-myc expression induces apoptosis after withdrawing survival factors, it is not clear if activation of the endogenous c-myc gene is an apoptotic signal after toxicant exposure. The renal tubular epithelium is a target for many toxicants. c-myc expression is activated by tubular damage. In quiescent LLC-PK1 renal epithelial cells, c-myc but not max or mad mRNA is induced by the nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC). The kinetics of DCVC-induced c-myc expression and apoptosis suggested an association between cell death and prolonged activation of c-myc expression after toxicant exposure. Accordingly, prolonged activation of an estrogen receptor-Myc fusion construct, but not a construct in which a c-Myc transactivation domain had been deleted, was sufficient to induce apoptosis in LLC-PK1 cells. Moreover, under conditions in which necrosis was the predominant cell death pathway caused by DCVC in parental cells, overexpressing c-myc biased the cell death pathway toward apoptosis. DCVC also induced ornithine decarboxylase (odc) mRNA and activated the odc promoter. Activation of the odc promoter by DCVC required consensus c-Myc-Max binding sites in odc intron 1. Inhibiting ODC activity with alpha-difluoromethylornithine delayed DCVC-induced cell death. Therefore, odc is a target gene in the DCVC apoptotic pathway involving c-myc activation and contributes to apoptosis. Finally, a structurally related cytotoxic but nongenotoxic analog of DCVC did not induce c-myc and did not activate the odc promoter or induce apoptosis. The data support the hypothesis that activation of apoptotic cell death in quiescent renal epithelial cells involves induction of c-myc. This is the first study to demonstrate that c-myc induction by a specific nephrotoxicant leads to gene activation and cell death.     

Requirement for efficient interactions between CD4 and MHC class II molecules for survival of resting CD4+ T lymphocytes in vivo and for activation-induced cell death

Regulation of homeostasis in the immune system includes mechanisms that promote survival of resting T lymphocytes, and others that control activation-induced cell death (AICD). In this study, we report on the use of a transgenic mouse model to test the role of CD4-MHC class II interactions for the susceptibility of CD4+ T lymphocytes to AICD, and for the survival of resting CD4+ T cells in peripheral lymphoid organs. The only I-Abeta gene expressed in these mice is an Abetak transgene with a mutation that prevents MHC class II molecules from interacting with CD4. We show increased apoptosis in CD4+ T lymphocytes derived from wild-type, but not from mutant Abetak transgenic mice following stimulation with staphylococcal enterotoxin A. Therefore, AICD may be impaired in CD4+ T cells derived from mutant Abetak transgenic mice. Importantly, we observed much higher apoptosis in resting CD4+ T cells from mutant Abetak transgenic mice than from wild-type mice. Furthermore, resting CD4+ T cells from mutant Abetak transgenic mice expressed higher levels of cell surface CD95 (Fas, APO-1). Ab-mediated cross-linking of CD95 further increased apoptosis in CD4+ T cells from mutant Abetak transgenic mice, but not from wild-type mice, suggesting apoptosis involved CD95 signaling. When cocultured with APC-expressing wild-type MHC class II molecules, apoptosis in resting CD4+ T lymphocytes from mutant Abetak transgenic mice was reduced. Our results show for the first time that interactions between CD4 and MHC class II molecules are required for the survival of resting CD4+ T cells in peripheral lymphoid organs.     

Accelerated programmed cell death of MRL-lpr/lpr T lymphocytes.

MRL-lpr/lpr (lpr) mice develop a polyclonal accumulation of abnormal peripheral T lymphocytes, which bear surface alpha beta TCR, CD3, and the B220 isoform of CD45, but lack CD4, CD8, and CD2. These T cells have a constitutively phosphorylated CD3 zeta chain and manifest a defect in signal transduction that results in a lack of IL-2 production and proliferation. We investigated whether this signaling abnormality might contribute to their accumulation via a defect in T cell elimination in the periphery. T cell deletion occurs through a process of programmed cell death with DNA degradation, or apoptosis. Viable lymphocytes from lpr mice were found to undergo rapid programmed cell death in culture within 4 h without additional activation, which was not observed in lymphocytes from normal MRL-+/+ or C57BL/6-+/+ mice. Both nonmature B220+ and mature B220- T lymphocytes from lpr mice display this accelerated programmed cell death, indicating that this is a defect affecting all peripheral T lymphocytes in lpr mice. In vitro apoptosis of lpr T cells could be inhibited with PMA, a stimulator of protein kinase C. Thus, the massive accumulation of T lymphocytes in the lymphoid tissue of lpr mice is not due to a defect in their ability to undergo programmed cell death in vitro. The activation state of lpr T cells may contribute to their rapid degradation of DNA in vitro.     

IL-7 promotes CD95-induced apoptosis in B cells via the IFN-γ/STAT1 pathway

Interleukin-7 (IL-7) concentrations are increased in the blood of CD4+ T cell depleted individuals, including HIV-1 infected patients. High IL-7 levels might stimulate T cell activation and, as we have shown earlier, IL-7 can prime resting T cell to CD95 induced apoptosis as well. HIV-1 infection leads to B cell abnormalities including increased apoptosis via the CD95 (Fas) death receptor pathway and loss of memory B cells. Peripheral B cells are not sensitive for IL-7, due to the lack of IL-7Ra expression on their surface; however, here we demonstrate that high IL-7 concentration can prime resting B cells to CD95-mediated apoptosis via an indirect mechanism. T cells cultured with IL-7 induced high CD95 expression on resting B cells together with an increased sensitivity to CD95 mediated apoptosis. As the mediator molecule responsible for B cell priming to CD95 mediated apoptosis we identified the cytokine IFN-γ that T cells secreted in high amounts in response to IL-7. These results suggest that the lymphopenia induced cytokine IL-7 can contribute to the increased B cell apoptosis observed in HIV-1 infected individuals.     

p56Lck tyrosine kinase enhances the assembly of death-inducing signaling complex during Fas-mediated apoptosis

Although the death-inducing signaling complex (DISC) is rapidly assembled, several lines of evidence suggest that formation of this complex is not the first consequence of cell surface CD95 (Fas) stimulation but rather a later step in this process. Activation of Fas triggers a cascade of signaling events that culminate in cellular apoptosis. Tyrosine kinases are critical effectors in T cell activation. However, their functional involvement in death receptor-mediated apoptosis is unknown. Here, we used p56(Lck)-deficient cells to show that CD95-induced cell death is highly dependent on p56(Lck) activity and its localization within plasma membrane. We found that p56(Lck) acts upstream of the mitochondria; in the absence of p56(Lck), Bid cleavage and the release of cytochrome c were severely impaired. Moreover, p56(Lck)-deficient cells or cells expressing an inactive form of p56(Lck) displayed defective formation of the DISC post CD95 stimulation. In vivo reconstitution of thymocytes from p56(lck)-deficient mice, which are resistant to apoptosis, with p56(Lck) restored Fas-mediated cell death. Our results support a novel model whereby sensitivity to apoptosis is regulated through quantitative changes in the stoichiometry of DISC components triggered by p56(Lck) activation and localization.     

The relevance of NF-?B for CD95 Signaling in Tumor Cells

Most members of the death receptor family including CD95 (APO-1/Fas) have beenshown to induce both apoptosis as well as nonapoptotic pathways depending on thetissue and the circumstances. One of the nonapoptotic pathways emanating from CD95,activation of NF-?B, has recently been demonstrated to regulate invasiveness ofapoptosis resistant tumor cells. In contrast, activation of NF-?B in apoptosing cells isbelieved to be suppressed due to cleavage of various NF-?B pathway components byactive caspases that execute apoptosis. We now present data demonstrating that incertain highly CD95 apoptosis sensitive cells NF-?B is robustly activated. In factoverexpression of apoptosis inhibitors such as Bcl-2 or c-FLIPL in these cells results indecreased activation of NF-?B through CD95. We propose a model in which NF-?B isgenerally activated in certain cells but may have different functions depending onwhether cells are programmed to die or to survive.     

Differential regulation of cell cycle-related proteins by CD95 engagement in thymocytes and T cell leukemic cell line, Jurkat

CD95 engagement results in apoptosis in thymocytes and in the Jurkat human leukemic T cell line. Biochemical analyses in CD95-engaged thymocytes and Jurkat cells revealed dysregulation of the G1/S cell cycle control point. Cyclin E was upregulated upon CD95 engagement, suggesting G1-to-S progression, but there was no upregulation of cyclin A. Instead, cyclin E was degraded by caspases. In addition, c-myc that normally acts on S-phase progression through the activation of cdc25A appeared to be involved in the inhibition of S-phase progression after CD95 ligation. This implies that G1-->S progression and apoptosis are intimately linked in cells undergoing CD95 ligation. Furthermore, our data suggest that CD95-induced apoptosis occurs at the G1/S phase transition. We therefore suggest that CD95 engagement not only triggers death signals but also affects the G1/S checkpoint.     

CD30 signals integrate expression of cytotoxic effector molecules, lymphocyte trafficking signals, and signals for proliferation and apoptosis

Although CD30 has long been recognized as an important marker on many lymphomas of diverse origin and as activation molecule on B cells and T cells, its primary function has remained obscure. We now report that CD30 signals may serve to inhibit effector cell activity by integrating gene expression changes of several pathways important for cytotoxic NK and T cell effector function. In the large granular lymphoma line YT, CD30 signals down-regulate the expression of cytotoxic effector molecules, Fas ligand, perforin, granzyme B, and abrogate cytotoxicity. c-myc, a regulator of proliferation and an upstream regulator of Fas ligand expression, is completely suppressed by CD30. Furthermore, CD30 signals strongly induce CCR7, suggesting a role for CD30 signals in the homing of lymphocytes to lymph nodes. The up-regulation of Fas, death receptor 3, and TNF-related apoptosis-inducing ligand by CD30 indicates an increase in susceptibility to apoptotic signals whereas up-regulation of TNFR-associated factor 1 and cellular inhibitor of apoptosis 2 protect cells from certain types of apoptosis. Using gene microarrays, 750 gene products were induced and 90 gene products were suppressed >2-fold by CD30 signals. Signals emanating from CD30 use both TNFR-associated factor 2-dependent and -independent pathways. The integration of CD30 signals in a lymphoma line suggests that CD30 can down-modulate lymphocyte effector function and proliferation while directing the cells to lymph nodes and increasing their susceptibility to certain apoptotic signals. These studies may provide a molecular mechanism for the recently observed CD30-mediated suppression of CTL activity in vivo in a diabetes model.     

Protein kinase C inhibits CD95 (Fas/APO-1)-mediated apoptosis by at least two different mechanisms in Jurkat T cells.

We have recently reported that activation of protein kinase C (PKC) plays a negative role in CD95-mediated apoptosis in human T cell lines. Here we present data indicating that although the PKC-induced mitogen-activated protein kinase pathway could be partially implicated in the abrogation of CD95-mediated apoptosis by phorbol esters in Jurkat T cells, the major inhibitory effect is exerted through a PKC-dependent, mitogen-activated protein kinase-independent signaling pathway. Furthermore, we demonstrate that activation of PKC diminishes CD95 receptor aggregation elicited by agonistic CD95 Abs. On the other hand, it has been reported that UV radiation-induced apoptosis is mediated at least in part by the induction of CD95 oligomerization at the cell surface. Here we show that activation of PKC also inhibits UVB light-induced CD95 aggregation and apoptosis in Jurkat T cells. These results reveal a novel mechanism by which T cells may restrain their sensitivity to CD95-induced cell death through PKC-mediated regulation of CD95 receptor oligomerization at the cell membrane.     

Loss of drug-induced activation of the CD95 apoptotic pathway in a cisplatin-resistant testicular germ cell tumor cell line

Testicular germ cell tumors (TGCTs) are unusually sensitive to cisplatin. In the present study the role of the CD95 death pathway in cisplatin sensitivity of TGCT cells was studied in Tera and its in vitro acquired cisplatin-resistant subclone Tera-CP. Cisplatin induced an increase in CD95 membrane expression, which preceded the onset of apoptosis. Cisplatin-induced apoptosis was efficiently blocked by caspase-8 inhibitor zIETD-fmk in Tera cells, but only partially in Tera-CP cells. In addition, cisplatin induced FADD and caspase-8 recruitment to the CD95 receptor in Tera cells, which was not noticed in Tera-CP cells. Moreover, overexpression of vFLIP reduced apoptosis induction by cisplatin in Tera cells. CD95L-blocking experiments revealed the involvement of CD95/CD95L interactions in cisplatin-induced apoptosis of Tera cells as well as cisplatin-sensitive 833KE TGCT cells. Tera and 833KE cells, treated with low doses of cisplatin, were sensitive for an apoptosis-inducing anti-CD95 antibody. In contrast, CD95L blocking had no effect on cisplatin-induced apoptosis in Tera-CP or Scha, an intrinsic resistant TGCT cell line, nor did anti-CD95 antibody induce additional apoptosis in cisplatin-treated Tera-CP or Scha cells. Taken together, these results show that (1) cisplatin sensitivity of TGCT cells is dependent on the activation of the CD95 death pathway and (2) loss of cisplatin-induced activation of this CD95 signaling pathway may result in resistance to cisplatin.     

Fas-independent apoptosis in T-cell tumours induced by the CD2-myc transgene

Depending on the cellular context, the Myc oncoprotein is capable of promoting cell proliferation or death by apoptosis. These observations suggest that apoptosis in response to deregulated gene expression may represent a natural brake to tumour development. The pathways by which Myc induces apoptosis are as yet poorly characterised although recent observations on rat fibroblasts over-expressing Myc have demonstrated a requirement for the Fas pathway. To investigate the role of Fas in Myc-induced lymphomagenesis we backcrossed CD2-myc mice onto an lpr background. Rates of tumour development and phenotypic properties, including levels of apoptosis were indistinguishable from CD2-myc controls. Further, tumour cell lines derived from mice expressing a regulatable form of Myc showed inducible apoptosis at similar rates regardless of their lpr genotype. These results show that activation of c-myc and loss of Fas do not collaborate in T lymphoma development and that Myc-induced apoptosis in T-cells occurs by Fas-independent pathways.