Delaying mitotic exit downregulates FLIP expression and strongly sensitizes tumor cells to TRAIL.

Many of the current antitumor therapeutic strategies are based on the perturbation of the cell cycle, especially during mitosis. Antimitotic drugs trigger mitotic checkpoint activation, mitotic arrest and eventually cell death. However, mitotic slippage represents a major mechanism of resistance to these treatments. In an attempt to circumvent the process of slippage, targeting mitotic exit has been proposed as a better strategy to kill tumor cells. In this study, we show that treatments that induce mitotic checkpoint activation and mitotic arrest downregulate FLICE-like inhibitory protein (FLIP) levels and sensitize several tumor cell lines to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. Interestingly, we also demonstrate that in absence of mitotic checkpoint activation, mitotic arrest induced either by Cdc20 knockdown or overexpression of nondegradable cyclin B is sufficient to induce both FLIP downregulation and sensitivity to TRAIL. In summary, our data suggest that a combination of antimitotic drugs targeting cyclin B degradation and TRAIL might prevent mitotic slippage and allow tumor cells to reach the threshold for apoptosis induction, thereby facilitating tumor suppression.

Control of FLIP(L) expression and TRAIL resistance by the extracellular signal-regulated kinase1/2 pathway in breast epithelial cells.

Increased activation of the epidermal growth factor receptor (EGFR) is frequently observed in tumors, and inhibition of the signaling pathways originated in the EGFR normally renders tumor cells more sensitive to apoptotic stimuli. However, we show that inhibition of EGFR signaling in non-transformed breast epithelial cells by EGF deprivation or gefitinib, an inhibitor of EGFR tyrosine kinase, causes the upregulation of the long isoform of caspase-8 inhibitor FLICE-inhibitory protein (FLIP(L)) and makes these cells more resistant to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway plays a pivotal role in the regulation of FLIP(L) levels and sensitivity to TRAIL-induced apoptosis by EGF. Upregulation of FLIP(L) upon EGF deprivation correlates with a decrease in c-Myc levels and c-Myc knockdown by siRNA induces FLIP(L) expression. FLIP(L) upregulation and resistance to TRAIL in EGF-deprived cells are reversed following activation of an estrogen activatable form of c-Myc (c-Myc-ER). Finally, constitutive activation of the ERK1/2 pathway in HER2/ERBB2-transformed cells prevents EGF deprivation-induced FLIP(L) upregulation and TRAIL resistance. Collectively, our results suggest that a regulated ERK1/2 pathway is crucial to control FLIP(L) levels and sensitivity to TRAIL in non-transformed cells, and this mechanism may explain the increased sensitivity of tumor cells to TRAIL, in which the ERK1/2 pathway is frequently deregulated.

The therapeutic potential of TRAIL receptor signalling in cancer cells.

In tumour cells, activation of the apoptotic machinery by death receptor ligands of the tumour necrosis factor (TNF) receptor superfamily of cytokines represents a novel therapeutic strategy. However, systemic treatment of tumours with TNF-α and CD95 ligand may produce severe toxic effects. The tumour necrosis-related apoptosisinducing ligand (TRAIL) is a member of the TNF family capable of inducing apoptosis in a wide variety of cancer cells upon binding to pro-apoptotic receptors, while having no effect on the majority of normal human cells tested. Interestingly, preclinical studies in mice and nonhuman primates showed no systemic cytotoxicity upon injection of either recombinant TRAIL or agonistic TRAIL-receptor antibodies. Furthermore, these treatments have been shown to effectively suppress the growth of a range of tumour xenografts. Although unwanted effects of some TRAIL preparations have been reported in normal cells, the use of TRAIL receptor agonists could represent a suitable approach in cancer therapy. Here, we shall review our current understanding of apoptotic and non-apoptotic TRAIL signalling, the therapeutic potential of TRAIL-based approaches in cancer treatment, and the results of phase 1 and 2 clinical trials with recombinant TRAIL or agonistic TRAIL receptor antibodies, either as monotherapy or in combination with other chemotherapeutic agents.

The therapeutic potential of TRAIL receptor signalling in cancer cells

In tumour cells, activation of the apoptotic machinery by death receptor ligands of the tumour necrosis factor (TNF) receptor superfamily of cytokines represents a novel therapeutic strategy. However, systemic treatment of tumours with TNF-α and CD95 ligand may produce severe toxic effects. The tumour necrosis-related apoptosisinducing ligand (TRAIL) is a member of the TNF family capable of inducing apoptosis in a wide variety of cancer cells upon binding to pro-apoptotic receptors, while having no effect on the majority of normal human cells tested. Interestingly, preclinical studies in mice and nonhuman primates showed no systemic cytotoxicity upon injection of either recombinant TRAIL or agonistic TRAIL-receptor antibodies. Furthermore, these treatments have been shown to effectively suppress the growth of a range of tumour xenografts. Although unwanted effects of some TRAIL preparations have been reported in normal cells, the use of TRAIL receptor agonists could represent a suitable approach in cancer therapy. Here, we shall review our current understanding of apoptotic and non-apoptotic TRAIL signalling, the therapeutic potential of TRAIL-based approaches in cancer treatment, and the results of phase 1 and 2 clinical trials with recombinant TRAIL or agonistic TRAIL receptor antibodies, either as monotherapy or in combination with other chemotherapeutic agents (AU)

Mitotic arrest and JNK-induced proteasomal degradation of FLIP and Mcl-1 are key events in the sensitization of breast tumor cells to TRAIL by antimicrotubule agents.

Breast tumor cells are often resistant to tumor necrosis factor-related apoptosis-inducing ligand (tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)/APO-2 L). Here, we describe the sensitization by microtubule-interfering agents (MIAs) to TRAIL-induced apoptosis in breast tumor cells through a mitotic arrest and c-Jun N-terminal kinase (JNK)-dependent mechanism. MIA treatment resulted in BubR1-dependent mitotic arrest leading to the sustained activation of JNK and the proteasome-mediated downregulation of cellular FLICE-inhibitory protein (cFLIP) and myeloid cell leukemia-1 (Mcl-1) expression. The JNK inhibitor SP600125 abrogated MIA-induced mitotic arrest and downregulation of cFLIP and Mcl-1 and reduced the apoptosis caused by the combination of MIAs and TRAIL. Silencing of cFLIP and Mcl-1 expression by RNA interference resulted in a marked sensitization to TRAIL-induced apoptosis. Furthermore, in FLIP-overexpressing cells, MIA-induced sensitization to TRAIL-activated apoptosis was markedly reduced. In summary, our results show that mitotic arrest imposed by MIAs activates JNK and facilitates TRAIL-induced activation of an apoptotic pathway in breast tumor cells by promoting the proteasome-mediated degradation of cFLIP and Mcl-1.

The mitogen-activated protein kinase pathway can inhibit TRAIL-induced apoptosis by prohibiting association of truncated Bid with mitochondria.

Breast cancer cells often show increased activity of the mitogen-activated protein kinase (MAPK) pathway. We report here that this pathway reduces their sensitivity to death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and present the underlying mechanism. Activation of protein kinase C (PKC) inhibited TRAIL-induced apoptosis in a protein synthesis-independent manner. Deliberate activation of MAPK was also inhibitory. In digitonin-permeabilized cells, PKC activation interfered with the capacity of recombinant truncated (t)Bid to release cytochrome c from mitochondria. MAPK activation did not affect TRAIL or tumor necrosis factor (TNF)alpha-induced Bid cleavage. However, it did inhibit translocation of (t)Bid to mitochondria as determined both by subcellular fractionation analysis and confocal microscopy. Steady state tBid mitochondrial localization was prohibited by activation of the MAPK pathway, also when the Bcl-2 homology domain 3 (BH3) domain of tBid was disrupted. We conclude that the MAPK pathway inhibits TRAIL-induced apoptosis in MCF-7 cells by prohibiting anchoring of tBid to the mitochondrial membrane. This anchoring is independent of its interaction with resident Bcl-2 family members.

The differential sensitivity of Bc1-2-overexpressing human breast tumor cells to TRAIL or doxorubicin-induced apoptosis is dependent on Bc1-2 protein levels.

Bc1-2 protein is a potent anti-apoptotic protein that inhibits a mitochondria-operated pathway of apoptosis in many cells. DNA damaging agents and death receptor ligands can activate this mitochondrial apoptotic mechanism. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been suggested to escape from the inhibitory action of Bc1-2 protein. We show that in human breast tumor MCF-7 cells, TRAIL induced a mitochondrial pathway of apoptosis that involved cytochrome c release from mitochondria and activation of caspase 9. The DNA damaging drug doxorubicin also activated this mitochondria-regulated mechanism of apoptosis, which was inhibited in Bc1-2-overexpressing cells. We also demonstrate that in MCF-7 cells Bc1-2 might confer resistance to TRAIL-induced apoptosis, depending on the expression levels of the anti-apoptotic protein. These results indicate that enhanced expression of Bc1-2 in tumor cells can render these cells less sensitive not only to chemotherapeutic drugs but also to TRAIL.

Activation of protein kinase C inhibits TRAIL-induced caspases activation, mitochondrial events and apoptosis in a human leukemic T cell line.

TRAIL causes apoptosis in numerous types of tumor cells. However, the mechanisms regulating TRAIL-induced apoptosis remain to be elucidated. We have investigated the role of PKC in regulating TRAIL-induced mitochondrial events and apoptosis in the Jurkat T cell line. We found a caspase-dependent decline in mitochondrial membrane potential and translocation of cytochrome c from mitochondria into the cytosol in response to TRAIL. Both these events were prevented by PKC activation. Moreover, PKC activation considerably reduced the activation of caspases, PARP cleavage and apoptosis when induced upon TRAIL treatment. MAPK activation was involved in the mechanism of PKC-mediated inhibition of TRAIL-induced cytochrome c release from mitochondria. Furthermore, inhibition of the MAPK pathway partially reversed the PKC-mediated inhibition of TRAIL-induced apoptosis. Besides, PKC activation may also inhibit the TRAIL-induced apoptosis through a MAPK-independent mechanism. Altogether, these results indicate a negative role of PKC in the regulation of apoptotic signals generated upon TRAIL receptor activation.

Interferon-gamma sensitizes human myeloid leukemia cells to death receptor-mediated apoptosis by a pleiotropic mechanism.

The role of interferon (IFN)-gamma as a sensitizing agent in apoptosis induced by ligation of death receptors has been evaluated in human myeloid leukemia cells. Incubation of U937 cells with IFN-gamma sensitized these cells to apoptosis induced by tumor necrosis factor-alpha, agonistic CD95 antibody, and tumor necrosis factor-related apoptosis-inducing ligand. Other human myeloid leukemic cells were also sensitized by IFN-gamma to death receptor-mediated apoptosis. Treatment of U937 cells with IFN-gamma up-regulated the expression of caspase-8 and potently synergized with death receptor ligation in the processing of caspase-8 and BID cleavage. Concomitantly, a marked down-regulation of BCL-2 protein was also observed in cells incubated with IFN-gamma. Furthermore, the caspase-dependent generation of a 23-kDa fragment of BCL-2 protein, the release of cytochrome c from mitochondria and the activation of caspase-9 were also enhanced upon death receptor ligation in IFN-gamma-treated cells. Ectopically expressed Bcl-2 protein inhibited IFN-gamma-induced sensitization to apoptosis. In summary, these results indicate that IFN-gamma sensitizes human myeloid leukemic cells to a death receptor-induced, mitochondria-mediated pathway of apoptosis.

Apoptosis of haematopoietic cells upon thymidylate synthase inhibition is independent of p53 accumulation and CD95-CD95 ligand interaction.

Treatment of haematopoietic BA/F3 cells with the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FUdR) activated apoptosis through a mechanism that required continuous protein synthesis and was inhibited by Bcl-2 over-expression. Analysis of p53 levels in cells treated with FUdR indicated a marked accumulation of this protein. Accumulation of p53 was also observed in cells over-expressing Bcl-2. In BA/F3 cells transfected with a cDNA coding for the human papilloma virus protein E6, p53 accumulation after FUdR treatment was inhibited markedly. However, apoptosis was induced in both control and E6 cells to a similar extent. The role of the CD95/CD95 ligand (CD95L) system in FUdR-induced apoptosis was also assessed. As determined by reverse transcriptase PCR, BA/F3 expressed a low constitutive level of CD95L mRNA, which decreased following FUdR treatment. Moreover, blocking CD95-CD95L interactions with antagonistic CD95 monoclonal antibody did not prevent drug-induced apoptosis. Furthermore, analysis of caspase involvement showed important differences in apoptosis induced by CD95-triggering or FUdR treatment. In summary, these results suggest that apoptosis induced by thymineless stress in haematopoietic BA/F3 cells occurs by a mechanism that does not require accumulation of p53 and which is independent of CD95-CD95L interactions.

Interferon-gamma treatment elevates caspase-8 expression and sensitizes human breast tumor cells to a death receptor-induced mitochondria-operated apoptotic program.

In this report, we have assessed the role of IFN-gamma as a sensitizing agent in apoptosis mediated by activation of death receptor CD95 in breast tumor cells. Treatment of the tumor cell lines MCF-7 and MDA-MB231 with IFN-gamma significantly facilitated apoptosis induced by CD95 receptor ligation at the plasma membrane, independently of p53 status. In contrast, IFN-gamma treatment did not enhance the apoptotic effect of the DNA-damaging drug, doxorubicin. Analysis of apoptosis regulators indicated that caspase-8 mRNA and protein levels were up-regulated in both of the cell lines after treatment with IFN-gamma. Furthermore, IFN-gamma sensitized MCF-7 and MDA-MB231 cells to CD95-mediated activation of caspase-8, induction of cytochrome c release from mitochondria, and processing of caspase-9. Release of cytochrome c, caspases activation, and apoptosis were prevented in MCF-7 cells overexpressing Bcl-2. Altogether these results indicate that IFN-gamma, maybe through the elevation of caspase-8 levels, sensitizes human breast tumor cells to a death receptor-mediated, mitochondria-operated pathway of apoptosis.

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.

p53-mediated up-regulation of CD95 is not involved in genotoxic drug-induced apoptosis of human breast tumor cells.

Induction of CD95 (Fas/APO-1) and CD95 ligand during chemotherapeutic treatment may contribute to the death by apoptosis of some tumor cells. In this study, we have analyzed the role of the CD95 system in genotoxic drug-induced death of human breast tumor cells. Incubation of the breast tumor cell lines MCF-7 and EVSA-T with doxorubicin or methotrexate caused apoptosis after 48 h of treatment. These drugs induced a marked increase in the level of CD95 mRNA and protein in wild-type p53-expressing MCF-7 cells. On the contrary, the breast cancer cell line EVSA-T that expresses high levels of an inactive form of p53, did not up-regulate CD95 upon drug treatment. Elevation of CD95 expression by DNA-damaging drugs was notably blocked in MCF-7 cells expressing the human papillomavirus type 16 E6 protein (E6 cells) which prevented p53 accumulation upon DNA damage. However, E6 cells were still killed by the drugs. Furthermore, the genotoxic drugs did not induce the expression of CD95 ligand in MCF-7 cells at doses that caused apoptosis in these breast tumor cells. Moreover, drug-induced apoptosis of breast tumor cells was not prevented in the presence of either a CD95 antagonistic antibody or a CD95 ligand blocking antibody. We also observed a strong synergism between lower doses of DNA-damaging drugs and CD95 agonistic antibody in the induction of apoptosis in MCF-7 cells. In summary, our data indicate that drug-induced apoptosis of breast tumor cells occurs by a CD95/CD95L-independent mechanism although by elevating the tumor suppressor proteins p53 and CD95, genotoxic drugs may sensitize breast tumor cells to CD95-mediated apoptosis.

Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidification.

In order to determine whether disruption of mitochondrial function could trigger apoptosis in murine haematopoietic cells, we used the potassium ionophore valinomycin. Valinomycin induces apoptosis in the murine pre-B cell line BAF3, which cannot be inhibited by interleukin-3 addition or Bcl-2 over-expression. Valinomycin triggers rapid loss of mitochondrial membrane potential. This precedes cytoplasmic acidification, which leads to cysteine-active-site protease activation, DNA fragmentation and cell death. Bongkrekic acid, an inhibitor of the mitochondrial permeability transition, prevents acidification and subsequent induction of apoptosis by valinomycin.

Activation of protein kinase C attenuates early signals in Fas-mediated apoptosis.

Activation of protein kinase C (PKC) has been reported to inhibit Fas (APO-1, CD95)-mediated apoptosis in different cellular systems. Human Jurkat leukemic T cells express the Fas antigen in the cell membrane and undergo apoptosis upon cross-linking by anti-Fas monoclonal antibodies (mAb). Cleavage of the apoptosis-associated protease CPP32 and its substrate poly(ADP-ribose)polymerase are observed after the engagement of Fas antigen with mAb. In this report, we show that all these effects are substantially inhibited by the activation of PKC with a phorbol ester. Bisindolylmaleimide, an inhibitor of PKC, prevents phorbol ester-induced down-regulation of Fas signaling. Inhibition of Fas-mediated cell death by phorbol ester is also observed in other human leukemic T cell lines. Cross-linking of Fas antigen by mAb results in the rapid increase in tyrosine phosphorylation of several protein substrates which is further elevated in the presence of the protein tyrosine phosphatase inhibitor, orthovanadate. Furthermore, orthovanadate markedly enhances the cell death response to Fas mAb in different human leukemic T cell lines and human T cell blasts. These effects of orthovanadate on early tyrosine phosphorylation and cell death are clearly diminished by PKC activation. These results strongly suggest that tyrosine phosphorylation is involved in Fas signaling in apoptosis and that PKC plays a negative role in Fas-mediated apoptosis by counteracting at a very early stage the signals generated following cross-linking of this receptor.

Overexpression of a heterologous thymidine kinase delays apoptosis induced by factor deprivation and inhibitors of deoxynucleotide metabolism.

Perturbing deoxyribonucleoside triphosphate (dNTP) metabolism with inhibitors of the de novo synthesis of dNTP causes apoptosis in the interleukin-3 (IL-3)-dependent pre-B cell line BAF3. Under these conditions apoptosis is prevented when deoxyribonucleosides for dNTP synthesis are supplied in the culture medium. On the other hand, removal of IL-3 from cultures of BAF3 cells resulted in down-regulation of thymidine kinase activity, rapid imbalance in dNTP levels, and apoptosis. In this study we show that overexpression of a heterologous thymidine kinase, herpes simplex virus thymidine kinase (TK), in BAF3 cells protects these cells from apoptosis induced by either inhibitors of dNTP synthesis or IL-3 deprivation. This protection against apoptosis is abrogated by 9-(4-hydroxybutyl)-N2-phenylguanine, a specific inhibitor of herpes simplex virus-1 TK. These results suggest that deoxyribonucleoside kinases, particularly TK, may be important in the regulation of apoptosis in hemopoietic cells.

Intracellular acidification induces apoptosis by stimulating ICE-like protease activity.

ICE-like protease activation and DNA fragmentation are preceded by a decrease in intracellular pH (pHi) during apoptosis in the IL-3 dependent cell line BAF3. Acidification occurs after 7 hours in cells deprived of IL-3 and after 4 hours when cells are treated with etoposide, close to the time of detection of ICE-like protease activity. Increasing extracellular pH reduces ICE-like protease activation and DNA fragmentation. Bcl-2 over-expression both delays acidification and inhibits ICE-like protease activation. Generation of a rapid intracellular pH decrease, using the ionophore nigericin, induces ICE-like protease activation and apoptosis. ZVAD, a cell permeable inhibitor of ICE-like proteases, does not affect acidification but inhibits apoptosis induced by IL-3 removal or nigericin treatment. These data suggest that intracellular acidification triggers apoptosis by directly or indirectly activating ICE-like proteases.

dNTP pools imbalance as a signal to initiate apoptosis.

Fidelity in DNA synthesis and repair is largely dependent on a balanced supply of deoxynucleotide triphosphate (dNTP) pools. Results from different groups have shown that alterations in dNTP supply result in DNA fragmentation and cell death with characteristics of apoptosis. We have recently shown that in apoptosis driven by deprivation of interleukin-3 (IL-3) in a murine hemopoietic cell line, there is a rapid imbalance in the availability of dNTP that precedes DNA fragmentation. In these cells, dNTP pool balance is closely coupled to the function of the salvage pathway of dNTP synthesis. Apoptosis, induced by treatment of these cells with drugs that inhibit the de novo dNTP synthesis, is prevented when dNTP precursors are supplied through the salvage pathway. IL-3 regulates thymidine kinase activity, suggesting that alterations in dNTP metabolism after IL-3 deprivation could be a relevant event in the commitment of hemopoietic cells to apoptosis.

An apoptotic endonuclease activated either by decreasing pH or by increasing calcium.

DNA fragmentation in isolated nuclei from the murine IL3-dependent bone marrow cell line BAF3 could be stimulated either by decreasing pH below 6.5 or by adding microM calcium at neutral pH. An endonuclease which could also be stimulated either by a decrease in pH, to 6.5, or by the presence of microM calcium at neutral pH, was purified 10(4)-fold from nuclei of BAF3 cells. Digestion of DNA with the purified enzyme resulted in 5'-terminal hydroxyl and 3'-terminal phosphate ends. These characteristics are distinct from those described for other mammalian endonucleases. The possible role of this enzyme in genome digestion during apoptosis is discussed.

Reactive oxygen intermediate(s) (ROI): common mediator(s) of poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis.

HL-60 acute myeloblastic and U937 monoblastoid leukaemic cell lines both cleave poly(ADP-ribose)polymerase (PARP), at the onset of apoptosis, in response to a wide range of cytotoxic agents. This appears to be a common feature of leukaemic cell apoptosis. However, in the chronic myelogenous leukaemic (CML) derived cell line, K562, no such cleavage was detectable. This correlated with previous findings that this cell line is particularly resistant to apoptosis induced by cytotoxic agents. Proteolytic cleavage of PARP and the subsequent progression to apoptosis was inhibited by two protease inhibitors NEM and IOD. As both PARP cleavage and DNA fragmentation appeared closely linked in these cell lines, anti-oxidants (previously shown to be effective inhibitors of DNA fragmentation and apoptosis) were also demonstrated to prevent PARP cleavage. These results combine to suggest that ROI may mediate PARP cleavage, DNA fragmentation and the eventual apoptosis of these cells following cytotoxic insult.