Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome.

Much effort has been put in the discovery of ways to selectively kill p53-deficient tumor cells and targeting cell cycle checkpoint pathways has revealed promising candidates. Studies in zebrafish and human cell lines suggested that the DNA damage response kinase, checkpoint kinase 1 (Chk1), not only regulates onset of mitosis but also cell death in response to DNA damage in the absence of p53. This effect reportedly relies on ataxia telangiectasia mutated (ATM)-dependent and PIDDosome-mediated activation of Caspase-2. However, we show that genetic ablation of PIDDosome components in mice does not affect cell death in response to γ-irradiation. Furthermore, Chk1 inhibition largely failed to sensitize normal and malignant cells from p53(-/-) mice toward DNA damaging agents, and p53 status did not affect the death-inducing activity of DNA damage after Chk1 inhibition in human cancer cells. These observations argue against cross-species conservation of a Chk1-controlled cell survival pathway demanding further investigation of the molecular machinery responsible for cell death elicited by forced mitotic entry in the presence of DNA damage in different cell types and model organisms.

BH3-only protein Bmf mediates apoptosis upon inhibition of CAP-dependent protein synthesis.

Tight transcriptional regulation, alternative splicing and/or post-translational modifications of BH3-only proteins fine-tune their proapoptotic function. In this study, we characterize the gene locus of the BH3-only protein Bmf (Bcl-2-modifying factor) and describe the generation of two major isoforms from a common transcript in which initiation of protein synthesis involves leucine-coding CUG. Bmf(CUG) and the originally described isoform, Bmf-short, display comparable binding affinities to prosurvival Bcl-2 family members, localize preferentially to the outer mitochondrial membrane and induce rapid Bcl-2-blockable apoptosis. Notably, endogenous Bmf expression is induced on forms of cell stress known to cause repression of the CAP-dependent translation machinery such as serum deprivation, hypoxia, inhibition of the PI3K/AKT pathway or mTOR, as well as direct pharmacological inhibition of the eukaryotic translation initiation factor eIF-4E. Knock down or deletion of Bmf reduces apoptosis under some of these conditions, demonstrating that Bmf can act as a sentinel for stress-impaired CAP-dependent protein translation machinery.

The BCL2 rheostat in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia.

Glucocorticoid (GC)-induced apoptosis is essential in the treatment of acute lymphoblastic leukemia (ALL) and related malignancies. Pro- and anti-apoptotic members of the BCL2 family control many forms of apoptotic cell death, but the extent to which this survival 'rheostat' is involved in the beneficial effects of GC therapy is not understood. We performed systematic analyses of expression, GC regulation and function of BCL2 molecules in primary ALL lymphoblasts and a corresponding in vitro model. Affymetrix-based expression profiling revealed that the response included regulations of pro-apoptotic and, surprisingly, anti-apoptotic BCL2 family members, and varied among patients, but was dominated by induction of the BH3-only molecules BMF and BCL2L11/Bim and repression of PMAIP1/Noxa. Conditional lentiviral gene overexpression and knock-down by RNA interference in the CCRF-CEM model revealed that induction of Bim, and to a lesser extent that of BMF, was required and sufficient for apoptosis. Although anti-apoptotic BCL2 members were not regulated consistently by GC in the various systems, their overexpression delayed, whereas their knock-down accelerated, GC-induced cell death. Thus, the combined clinical and experimental data suggest that GCs induce both pro- and anti-apoptotic BCL2 family member-dependent pathways, with the outcome depending on cellular context and additional signals feeding into the BCL2 rheostat.

G1 arrest by p16INK4A uncouples growth from cell cycle progression in leukemia cells with deregulated cyclin E and c-Myc expression.

The cell cycle inhibitor p16(INK4A) is frequently inactivated in acute lymphoblastic T-cell leukemia (T-ALL). We analyzed mechanisms and consequences of p16(INK4A) reconstitution in T-ALL cells lacking this tumor suppressor. CCRF-CEM cells with tetracycline-regulated p16(INK4A) expression underwent stable G1-phase cell cycle arrest for 72 h followed by massive apoptosis. p16(INK4A) expression caused pRB hypophosphorylation and repression of certain E2F target genes. Interestingly, cyclin E and c-Myc were not affected, suggesting pRB/E2F-independent expression of these E2F targets. Cyclin E/CDK2, however, was inactive due to stabilization and redistribution of p27(Kip1) from CDK4/CDK6 to CDK2. Analyses of c-Myc target genes suggested that c-Myc was transcriptionally inactive, which correlated with hypophosphorylation of the c-Myc inhibitor p107. Thus, p16(INK4A), although unable to repress the expression of deregulated cyclin E and c-Myc, functionally inactivated these potential oncogenes. p16(INK4A)-arrested cells showed morphologic changes, induction of T-cell-specific surface markers and repression of telomerase activity, suggesting differentiation. Moreover, p16(INK4A) reconstitution was associated with increased cellular volume, normal protein synthesis rates and elevated ATP levels. Taken together, p16(INK4A) reconstitution in p16(INK4A)-deficient T-ALL cells induced cell cycle arrest in the presence of cyclin E and c-Myc expression, uncoupled growth from cell cycle progression and caused a sequential process of growth, differentiation and apoptosis.

Glucocorticoid receptor heterozygosity combined with lack of receptor auto-induction causes glucocorticoid resistance in Jurkat acute lymphoblastic leukemia cells.

Glucocorticoids (GC) induce apoptosis in malignant lymphoblasts, but the mechanism of this process as well as that of the clinically important GC resistance is unknown. We investigated GC resistance in Jurkat T-ALL cells in which ectopic GC receptor (GR) restores GC sensitivity, suggesting deficient GR expression. Jurkat cells expressed one wild-type and one mutated (R477H) GR allele. GR(R477H) ligand-binding-dependent nuclear import, as revealed by live-cell microscopy of YFP-tagged GR, was unaffected. Transactivation and transrepression were markedly impaired; however, GR(R477H) did not act in a dominant-negative manner, that is, did not prevent cell death, when introduced into a GC-sensitive cell line by retroviral gene transfer. Contrary to another GR heterozygous, but GC-sensitive, T-ALL model (CCRF-CEM), Jurkats expressed lower basal GR levels and did not auto-induce their GR, as revealed by 'real-time' RT-PCR and immunoblotting. Absent GR auto-induction could not be restored by transgenic GR and, hence, was not caused by reduced basal GR levels. Thus, inactivation of one GR gene results in haploinsufficiency if associated with lack of GR auto-induction.

Cyclin D3 and c-MYC control glucocorticoid-induced cell cycle arrest but not apoptosis in lymphoblastic leukemia cells.

Glucocorticoids (GC) induce cell cycle arrest and apoptosis in lymphoblastic leukemia cells. To investigate cell cycle effects of GC in the absence of obscuring apoptotic events, we used human CCRF-CEM leukemia cells protected from cell death by transgenic bcl-2. GC treatment arrested these cells in the G1 phase of the cell cycle due to repression of cyclin D3 and c-myc. Cyclin E and Cdk2 protein levels remained high, but the kinase complex was inactive due to increased levels of bound p27(Kip1). Conditional expression of cyclin D3 and/or c-myc was sufficient to prevent GC-induced G1 arrest and p27(Kip1) accumulation but, importantly, did not interfere with the induction of apoptosis. The combined data suggest that repression of both, c-myc and cyclin D3, is necessary to arrest human leukemia cells in the G1 phase of the cell division cycle, but that neither one is required for GC-induced apoptosis.

The cell cycle inhibitor p16(INK4A) sensitizes lymphoblastic leukemia cells to apoptosis by physiologic glucocorticoid levels.

The cyclin-dependent kinase inhibitor p16(INK4A) is frequently inactivated in childhood T-cell acute lymphoblastic leukemia. To investigate possible consequences of this genetic alteration for tumor development, we conditionally expressed p16(INK4A) in the T-cell acute lymphoblastic leukemia line CCRF-CEM, which carries a homozygous deletion of this gene. In agreement with its reported function, p16(INK4A) expression was associated with hypophosphorylation of the retinoblastoma protein pRB and stable cell cycle arrest in G(0)/G(1), documenting that the pRB/E2F pathway is functional in these cells. Unexpectedly, p16(INK4A) expression increased the sensitivity threshold for glucocorticoid (GC)-induced apoptosis from therapeutic to physiologic levels. As a possible explanation for this phenomenon, we found that p16(INK4A)-arrested cells had elevated GC receptor expression associated with enhanced GC-mediated transcriptional activity and increased responsiveness of the GC-regulated cyclin D3 gene. These data are supported by our previous findings that GC receptor levels critically influence GC sensitivity and imply that p16(INK4A) inactivation, in addition to allowing unrestricted proliferation, represents a mechanism by which lymphoid tumor cells might escape cell death triggered by endogenous GC.

Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint.

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.

The cell cycle inhibitor p16(INK4A) sensitizes lymphoblastic leukemia cells to apoptosis by physiologic glucocorticoid levels.

The cyclin-dependent kinase inhibitor p16(INK4A) is frequently inactivated in childhood T-cell acute lymphoblastic leukemia. To investigate possible consequences of this genetic alteration for tumor development, we conditionally expressed p16(INK4A) in the T-cell acute lymphoblastic leukemia line CCRF-CEM, which carries a homozygous deletion of this gene. In agreement with its reported function, p16(INK4A) expression was associated with hypophosphorylation of the retinoblastoma protein pRB and stable cell cycle arrest in G(0)/G(1), documenting that the pRB/E2F pathway is functional in these cells. Unexpectedly, p16(INK4A) expression increased the sensitivity threshold for glucocorticoid (GC)-induced apoptosis from therapeutic to physiologic levels. As a possible explanation for this phenomenon, we found that p16(INK4A)-arrested cells had elevated GC receptor expression associated with enhanced GC-mediated transcriptional activity and increased responsiveness of the GC-regulated cyclin D3 gene. These data are supported by our previous findings that GC receptor levels critically influence GC sensitivity and imply that p16(INK4A) inactivation, in addition to allowing unrestricted proliferation, represents a mechanism by which lymphoid tumor cells might escape cell death triggered by endogenous GC.

Transplantation of IL-2-transduced murine bone marrow is associated with dose-dependent toxicity.

OBJECTIVE: The purpose of this study was to investigate the effects of interleukin-2 (IL-2) gene-transduced hematopoietic progenitor cells or cytotoxic function and systemic toxicity following syngeneic bone marrow transplantation. MATERIAL AND METHODS: Marrow of 5-fluorouracil pretreated donor mice were transfected with a retroviral vector containing the murine IL-2 gene and transplanted into lethally irradiated syngeneic hosts. RESULTS: Productive insertion of the IL-2 gene could be demonstrated at various intervals post-transplant without impairment of hematopoietic engraftment. Endogenously augmented IL-2 release resulted in a selective increase in CD4(+), CD8(+), and NK1.1(+) population in spleen and bone marrow, as well as significant cytolytic activity against syngeneic leukemia cells in vitro. Our results also illustrate the interdependence among the magnitude of systemic IL-2 levels, the number of IL-2-transduced cells in the transplant inoculum, and the appearance of systemic toxicity. Infusion of marrow transduced with high-titer, high-expressing IL-2 retrovirus resulted in significant morbidity and mortality in the recipients. Our studies demonstrate that mortality was secondary to severe lymphocytic infiltration of liver and lung, which was associated with increased expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1. Reducing the number of IL-2-transduced cells in the bone marrow inoculum, however, resulted in significantly improved survival with no adverse events being evident during the post-transplant period. CONCLUSION: Delivery of IL-2 to the bone marrow can be achieved by transplantation of genetically modified hematopoietic cells, however, the overall feasibility is strongly influenced by the number of transduced cells in the bone marrow inocolum and/or the expression pattern of IL-2 in vivo.

Identification of XDRP1; a Xenopus protein related to yeast Dsk2p binds to the N-terminus of cyclin A and inhibits its degradation.

Using the N-terminus of cyclin A1 in a two-hybrid screen as a bait, we identified a Xenopus protein, XDRP1, that contains a ubiquitin-like domain in its N-terminus and shows significant homology in its C-terminal 50 residues to Saccharomyces cerevisiae Dsk2 and Schizosaccharomyces pombe dph1. XDRP1 is a nuclear phosphoprotein in Xenopus cells, and its phosphorylation is mediated by cyclin A-dependent kinase. XDRP1 binds to both embryonic and somatic forms of cyclin A (A1 and A2) in Xenopus cells, but not to B-type cyclins. The N-terminal ubiquitin-like domain of XDRP1, but not the C-terminal Dsk2-like domain, is required for interaction with cyclin A. XDRP1 requires residues 130-160 of cyclin A1 for efficient binding, which do not include the destruction box of cyclin A. The addition of bacterially expressed XDRP1 protein to frog egg extract inhibited the Ca(2+)-induced degradation of cyclin A, but not that of cyclin B. The injection of XDRP1 protein into fertilized Xenopus eggs blocked embryonic cell division.

c-Myc does not prevent glucocorticoid-induced apoptosis of human leukemic lymphoblasts.

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. The signal transduction pathways leading to this clinically-relevant form of apoptosis have, however, not been sufficiently elucidated. GC bind to their specific receptor, a ligand-activated transcription factor of the Zn-finger type, that activates or represses transcription of GC-responsive genes. Previous studies in leukemia cells suggested that transcriptional repression of c-myc expression might be the crucial event in GC-induced apoptosis, although in other systems, c-Myc apparently increased the sensitivity to cell-death inducers. To address this controversy, we stably transfected the GC-sensitive human T-ALL cell line CEM-C7H2 with constructs allowing tetracycline-regulated expression of c-Myc. Subsequent analyses of these cell lines showed that overexpression of c-Myc per se had little, if any, effect on cell viability, although it rendered the cells more sensitive to apoptosis induced by low serum, confirming the functionality of the expressed transgene. More importantly, however, when the cells were treated with GC in the presence of exogenous c-Myc, they underwent apoptosis exceeding that in cells treated in the absence of transgenic c-Myc. The data indicate that c-myc downregulation is not critical for induction of cell-death by GC in this system, and support the notion that c-Myc sensitizes cells to apoptosis-inducing agents.

Sequence-specific transcription factors during glucocorticoid-induced apoptosis in acute lymphoblastic leukemia cells.

Glucocorticoids (GC) are known to induce programmed cell death (apoptosis) in certain hematologic malignancies, but the molecular basis of this clinically significant phenomenon is poorly understood. GC act via binding to their specific receptor, a ligand-activated transcription factor, and might induce apoptosis by transcriptional activation of "death" or repression of "survival" genes. GC regulate gene expression directly, i.e. via GC responsive elements, or indirectly by modulating the activity of other transcription factors such as AP-1, NF-KB, Oct, Ets, and CREB. To analyze possible alterations in the activity of these transcription factors during GC-induced apoptosis, we performed electrophoretic mobility shift assays using the human acute T-cell leukemia line CCRF-CEM C7H2 as a model system. Although AP-1 was highly inducible by phorbol ester treatment, it was almost undetectable in logarithmically growing cells and apparently unregulated during GC-induced apoptosis. Thus, alterations in AP-1 activity do not appear to be involved in GC-induced apoptosis. Oct, Ets, and CREB DNA binding activity were detectable prior to and during GC treatment, and appeared to have been down-regulated after 48 hours. At this time, however, cells had already undergone considerable apoptosis, and this downregulation might reflect cell death-associated protein degradation. In contrast, NF-KB DNA binding activity was reduced 12 to 24 hours after GC exposure but reached levels equal to or higher than pre-treatment levels after 48 hours. Thus, while AP-1, Oct, Ets, and CREB may not be involved in GC-induced apoptosis, the maintenance of NF-KB levels suggests that it may participate in this form of cell death.

Detection and quantitation of genetically marked acute myeloid leukemia by competitive polymerase chain reaction after autologous bone marrow transplantation: a preclinical model for minimal residual disease.

Preclinical models and methods aimed at detecting and quantitating minimal residual disease (MRD) after autologous bone marrow transplantation (BMT) for acute myeloid leukemia (AML) could facilitate assessment of innovative therapeutic strategies for their antileukemic potential. Among the various techniques exploited to identify MRD, polymerase chain reaction (PCR) proved to be a valuable tool in instances in which clonogeneic markers are involved during the evolution of disease. In human AML, however, detection of MRD by PCR is limited to a minority of subgroups, as clonospecific markers are absent or presently unknown. Although gene labeling has proved to be efficient in detecting marker-devoid leukemia cells in preclinical models, detection and quantitation by PCR have not yet been considered. We therefore developed an experimental model in which detection and quantitation of genetically marked murine AML cells are based on a highly sensitive two-step nested PCR and competitive PCR protocol, respectively. We further demonstrated its applicability to a murine syngeneic BMT model that was designed to monitor minimal numbers of gene-tagged AML cells at various time intervals after transplantation. Our results showed that detection and quantitation could reproducibly be achieved at levels as low as one in 10(6) and 10(5) cells, respectively.

Bcl-2 interferes with the execution phase, but not upstream events, in glucocorticoid-induced leukemia apoptosis.

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. Cell death is associated with activation of members of the interleukin-1beta-converting enzyme (ICE) protease/caspase family and, is presumably prevented by the anti-apoptotic protein Bcl-2. To further address the role of Bcl-2 in GC-mediated cytotoxicity, we generated subclones of the GC-sensitive human T-cell acute lymphoblastic leukemia line CCRF-CEM, in which transgenic Bcl-2 expression is regulated by tetracycline. Up to about 48 h, exogenous Bcl-2 almost completely protected these cells from apoptosis, digestion of poly-ADP ribose polymerase (PARP) and generation of Asp-Glu-Val-Asp cleaving (DEVDase) activity. However, when the cells were cultured for another 24 h in the continuous presence of GC, they underwent massive apoptosis that was associated with DEVDase activity and PARP cleavage. Bcl-2 did not markedly affect GC-mediated growth arrest, thereby separating the anti-proliferative from the apoptosis-inducing effect of GC. Moreover, Bcl-2 did not prevent the dramatic reduction in the levels of several mRNAs observed during GC treatment, including the transgenic Bcl-2 mRNA. Thus, Bcl-2 can be placed upstream of effector caspase activation, but downstream of other GC-regulated events, such as growth arrest and the potentially critical repression of steady state levels of multiple mRNA.

Irradiation induces G2/M cell cycle arrest and apoptosis in p53-deficient lymphoblastic leukemia cells without affecting Bcl-2 and Bax expression.

The tumor suppressor p53 has been implicated in gamma irradiation-induced apoptosis. To investigate possible consequences of wild-type p53 loss in leukemia, we studied the effect of a single dose of gamma irradiation upon p53-deficient human T-ALL (acute lymphoblastic leukemia) CCRF - CEM cells. Exposure to 3 - 96 Gy caused p53-independent cell death in a dose and time-dependent fashion. By electron microscopic and other criteria, this cell death was classified as apoptosis. At low to intermediate levels of irradiation, apoptosis was preceded by accumulation of cells in the G2/M phase of the cell division cycle. Expression of Bcl-2 and Bax were not detectably altered after irradiation. Expression of the temperature sensitive mouse p53 V135 mutant induced apoptosis on its own but only slightly increased the sensitivity of CCRF - CEM cells to gamma irradiation. Thus, in these, and perhaps other leukemia cells, a p53- and Bcl-2/Bax-independent mechanism is operative that efficiently senses irradiation effects and translates this signal into arrest in the G2/M phase of the cell cycle and subsequent apoptosis.

p53-induced apoptosis in the human T-ALL cell line CCRF-CEM.

The tumor suppressor p53 has been implicated in apoptosis induction and is mutated in human T-ALL CCRF-CEM cells. To investigate possible consequences of wild-type p53 loss, we reconstituted CEM-C7H2, a subclone of CCRF-CEM, with a temperature-sensitive p53 allele (p53ts). Stably transfected lines expressed high levels of p53ts and shift to the permissive temperature (32 degrees C) caused rapid induction of p53-regulated genes, such as p21(CIP1/WAF1), mdm-2 and bax. This was followed by extensive apoptosis within 24 h to 36 h, supporting the notion that mutational p53 inactivation contributed to the malignant phenotype. p53-dependent apoptosis was preceded by digestion of poly(ADP-ribose) polymerase, a typical target of interleukin-1beta-converting enzyme (ICE)-like proteases/caspases, and was markedly resistant to the ICE/caspase-1 and FLICE/caspase-8 inhibitor acetyl-Tyr-Val-Ala-Asp.chloromethylketone (YVAD), but sensitive to the CPP32/caspase-3 inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp.fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (zVAD), a caspase inhibitor with broader specificity. This indicated an essential involvement of caspases, but argued against a significant role of ICE/caspase-1 or FLICE/caspase-8. Actinomycin D or cycloheximide prevented cell death, suggesting that, in this system, p53-induced apoptosis depends upon macromolecule biosynthesis. Introduction of functional p53 into CEM cells enhanced their sensitivity to the DNA-damaging agent doxorubicin, but not to the tubulin-active compound vincristine. Thus, mutational p53 inactivation in ALL might entail relative resistance to DNA-damaging, but not to tubulin-destabilizing, chemotherapy.

Drug-induced apoptosis is associated with enhanced Fas (Apo-1/CD95) ligand expression but occurs independently of Fas (Apo-1/CD95) signaling in human T-acute lymphatic leukemia cells.

Induction of apoptosis is considered to be the underlying mechanism that accounts for the efficiency of chemotherapeutic drugs. It has recently been proposed that induction of Fas ligand (FasL) expression with subsequent autocrine and/or paracrine induction of cell death through binding to the Fas (Apo-1/CD95) membrane accounts for chemotherapy-associated apoptosis. In the present study, we analyzed the significance of FasL expression in the mediation of drug-induced apoptosis in the T-acute lymphatic leukemia model CEM. In particular, we examined the potential of the tumor drugs fludarabine, doxorubicin, and cisplatin to induce FasL expression. We also raised the question of whether apoptosis induced by these drugs occurs through the Fas pathway and hence can be blocked by the cowpox virus protein CrmA, a specific inhibitor of this pathway. All tumor drugs examined led to an increase in FasL protein. However, overexpression of CrmA had no effect on drug-induced apoptosis. Moreover, neither incubation with inhibitory monoclonal antibodies against Fas that completely prevented Fas-induced apoptosis in these cells nor pretreatment with a monoclonal antibody to FasL affected drug-induced cell death. Our observations suggest a Fas/FasL-independent mechanism for drug-induced apoptosis and exclude the involvement of caspase 1 and caspase 8 in this process in T-acute lymphatic leukemia cells.

The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis.

According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1beta converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (DeltaPsim). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the DeltaPsim is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the DeltaPsim collapse and subsequent apoptosis. Cytosols from anti-Fas-treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their DeltaPsim. In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

Constitutive expression of Fas (Apo-1/CD95) ligand on multiple myeloma cells: a potential mechanism of tumor-induced suppression of immune surveillance.

The Fas (Apo-1/CD95) ligand (FasL) plays a central role in the elimination of target cells by effector T lymphocytes and in the suppression of cellular immune responses against nonmalignant and malignant cells. We show the expression of FasL on the surface of neoplastic plasma cells. We provide evidence that the FasL is functionally active because five of five neoplastic plasma cell lines tested killed CEM-C7H2 T-acute lymphoblastic leukemia (T-ALL) cells. The effect was mediated via the Fas (Apo-1/CD95) receptor molecule because blocking of Fas on the target cells or the FasL on the tumor cells by receptor- and ligand-specific monoclonal antibodies (MoAbs), respectively, protected T cells from being killed by myeloma cells. In addition, overexpression of the cowpox virus protein CrmA, a molecule with inhibitory potential on caspase-1 and caspase-8, specifically involved in Fas-induced signaling, protected T cells from being destroyed by the neoplastic cells or the agonistic anti-Fas MoAb. The potential of the malignant plasma cells to extinguish target T cells was independent of their own sensitivity to the agonistic anti-Fas MoAb, and FasL-positive (FasL+) CEM-C7H2 T cells were incapable of killing myeloma cells. Our results suggest that tumor cell-induced suppression of the immune system may be exerted via the FasL active on malignant plasma cells. Furthermore, loss of Fas expression or insensitivity to the agonistic anti-Fas MoAb do not seem to be prerequisites for myeloma cells to defeat T cells via Fas/FasL interaction.