p27Kip1 induces drug resistance by preventing apoptosis upstream of cytochrome c release and procaspase-3 activation in leukemic cells.

The cyclin-dependent kinase inhibitor p27Kip1 has been implicated as a drug resistance factor in tumor cells grown as spheroids or confluent monolayers. Here, we show that p27Kip1 overexpression also induces resistance to drug-induced apoptosis and cytotoxicity in human leukemic cells growing in suspension. The anti-apoptotic effect of p27Kip1 is not restricted to DNA-damaging agents but extends to the tubulin poison vinblastin, agonistic anti-Fas antibodies and macromolecule synthesis inhibitors. To further identify at which level this protein interferes with the cell death pathway, we investigated its influence on caspase activation and mitochondrial changes. Exposure of mock-transfected U937 cells to 50 microm etoposide activates procaspase-3 and the long isoform of procaspase-2 and induces mitochondrial potential decrease and cytochrome c release from mitochondria to the cytosol. All these events are prevented by p27Kip1 overexpression. p27Kip1 does not modulate Bcl-2, Bcl-X(L), Mcl-1 and Bax protein level in leukemic cells but suppresses Mcl-1 expression decrease observed in mock-transfected U937 cells undergoing etoposide-induced cell death. We conclude that p27Kip1 prevents cell death upstream of the final pathway common to many apoptotic stimuli that involves cytochrome c release from mitochondria and activation of downstream caspases.

Caspase-induced proteolysis of the cyclin-dependent kinase inhibitor p27Kip1 mediates its anti-apoptotic activity.

The caspase-mediated cleavage of a limited number of cellular proteins is a common feature of apoptotic cell death. This cleavage usually inhibits the function of the target protein or generates peptides that actively contribute to the death process. In the present study, we demonstrate that the cyclin-dependent kinase inhibitor p27Kip1 is cleaved by caspases in human leukemic cells exposed to apoptotic stimuli. We have shown recently that p27Kip1 overexpression delayed leukemic cell death in response to cytotoxic drugs. In transient transfection experiments, the p23 and the p15 N-terminal peptides generated by p27Kip1 proteolysis demonstrate an anti-apoptotic effect similar to that induced by the wild-type protein, whereas cleavage-resistant mutants have lost their protective effect. Moreover, stable transfection of a cleavage-resistant mutant of p27Kip1 sensitizes leukemic cells to drug-induced cell death. Altogether, these results indicate that proteolysis of p27Kip1 triggered by caspases mediates the anti-apoptotic activity of the protein.

Selective inhibition of apoptosis by TPA-induced differentiation of U937 leukemic cells.

U937 leukemic cells treated for 24 h with 16 nM 12-O-tetradecanoylphorbol 13-acetate (TPA), that induces their macrophagic terminal differentiation, become resistant to etoposide-induced apoptosis. Exposure of undifferentiated U937 cells to 50 microM etoposide for 6 h, that triggers apoptosis in 80% cells, activates procaspase-2L, -3 and -8, induces the mitochondrial release of cytochrome c and decreases Mcl-1 expression without modifying Bcl-2, Bcl-xL and Bax protein levels. All these events are inhibited in TPA-differentiated U937 cells that are also resistant to vinblastine-induced and Fas-mediated cell death. Interestingly, these cells are not inherently resistant to apoptosis induction. Exposure of TPA-differentiated U937 cells to 0.8 microg/ml cycloheximide for 24 h, that triggers apoptosis in 50% cells, activates procaspase-2L, -3 and -8, induces the mitochondrial release of cytochrome c and decreases Bcl-xL expression without modifying Bcl-2, Mcl-1 and Bax protein levels. All these events are not observed in undifferentiated cells treated in similar conditions. These results indicate that the apoptotic pathway that involves the release of cytochrome c from mitochondria and the cleavage of procaspases remains functional in TPA-differentiated cells.

Expression and activity of caspases 1 and 3 in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are characterized by abnormal growth of committed progenitors in clonogenic assay, with reduced number of colonies and decreased colony/cluster ratio. It has been suggested that excessive apoptosis is the cause of marrow failure in MDS. We studied the expression of caspase-1 (interleukin-1beta-converting enzyme, ICE) and caspase-3 (CPP32/apopain) in marrow mononuclear cells, and the growth pattern of committed progenitors in a series of 83 MDS cases. The percentage of apoptotic cells as detected by TUNEL technique, and the percentage of caspase-3-positive cells were significantly higher in refractory anemia (RA) and RA with ringed sideroblasts (RAS) than in chronic myelomonocytic leukemia (CMML), refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T). Spontaneous growth of CFU-GM was associated with a higher percentage of blasts, and with a lower expression of caspase-3 and caspase-1. The yield of CFU-E, BFU-E, and CFU-GM (in the presence of growth factors) was decreased by comparison to normal marrow, but large individual differences were observed in all cytological categories. Inhibition of caspase-1 and caspase-3 activities by specific inhibitors resulted in a significant increase of the production of all types of colonies (up to 50-fold of control). In the presence of caspase-3 inhibitor, the number of BFU-E and CFU-E was in the range of normal values in most cases of RA and RAS. In addition, caspase-1 and -3 protease activities were detectable by fluorogenic assay in all cases studied. Western blot analysis confirmed the expression of caspase-3, including the cleaved (activated)-p17 form in most cases of RA/RAS analyzed. It is concluded that caspase-3 is implicated in the increased apoptosis observed in MDS and that inhibition of its activity can restore at least partially the growth of committed progenitors.

Modulation of apoptotic pathways triggered by cytotoxic agents.

Anticancer drugs can induce tumour cell death by apoptosis. The main pathway from specific damage induced by the drug to apoptosis involves activation of caspases in the cytosol by pro-apoptotic molecules such as cytochrome c released from the mitochondria. At least in some cell types, anticancer drugs also upregulate the expression of death receptors and sensitize tumour cells to their cognate ligands, which could be used to amplify the response to cytotoxic drugs. The Bcl-2 family of proteins, which includes anti- and pro-apoptotic molecules, regulates cell sensitivity at the mitochondrial level. Chemotherapeutic drugs modulate their expression (e.g. through p53-dependent gene transcription), their activity (e.g. by phosphorylation) and their subcellular localization (e.g. by translocation of pro-apoptotic proteins from the cytosol to the mitochondria). When interacting with tumour cells, anticancer drugs also activate lipid- and kinase-dependent signalling pathways that modulate the death response to specific damage. Protective pathways include activation of NF kappa B transcription factor, accumulation of heat shock proteins and activation of proteins involved in cell cycle regulation. The recent identification on these pathways to cell death has suggested several new strategies to improve the therapeutic efficacy of currently used anticancer drug regimens.

BCR-ABL delays apoptosis upstream of procaspase-3 activation.

The p210(bcr-abl) protein was shown to inhibit apoptosis induced by DNA damaging agents. Apoptotic DNA fragmentation is delayed in the bcr-abl+ K562 and KCL-22 compared with the bcr-abl- U937 and HL-60 cell lines when treated with etoposide concentrations that induce similar DNA damage in the four cell lines. By the use of a cell-free system, we show that nuclei from untreated cells that express p210(bcr-abl) remain sensitive to apoptotic DNA fragmentation induced by triton-soluble extracts from p210(bcr-abl-) cells treated with etoposide. In the four tested cell lines, apoptotic DNA fragmentation is associated with a decreased expression of procaspase-3 (CPP32/Yama/apopain) and its cleavage into a p17 active fragment, whereas the long isoform of procaspase-2 (ICH-1L) remains unchanged and the poly(adenosine diphosphate-ribose)polymerase protein is cleaved. These events are delayed in bcr-abl+ compared with bcr-abl- cell lines. The role of p210(bcr-abl) in this delay is confirmed by comparing the effect of etoposide on the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent UT7 cells and the bcr-abl-transfected GM-CSF-independent UT7/9 clone. We conclude that the cytosolic pathway that leads to apoptotic DNA fragmentation in etoposide-treated leukemic cells is delayed upstream of procaspase-3-mediated events in bcr-abl+ cell lines.

Involvement of caspase-2 long isoform in Fas-mediated cell death of human leukemic cells.

Engagement of the plasma membrane receptor Fas can induce apoptosis of leukemic cells. Signaling through Fas requires the formation of a death-inducing signaling complex (DISC) that involves the cytoplasmic domain of Fas, the adaptor molecule FADD/MORT-1, and procaspase-8. The present study investigated whether another caspase, known as procaspase-2L, played a role in Fas-mediated cell death. A series of human leukemic variant cells was derived by stable transfection with a CASP2L antisense construct (CASP2L/AS). Specific down-regulation of procaspase-2L decreased the sensitivity of these cells to apoptosis induced by an agonistic anti-Fas antibody (Ab, clone CH11), as determined by studying DNA fragmentation, chromatin condensation, and externalization of phosphatidylserine on the plasma membrane. In leukemic cells transfected with an empty vector, anti-Fas Ab treatment activated caspase-8, decreased the expression of the BH3 domain-only protein Bid, triggered the release of cytochrome c from the mitochondria to the cytosol, and activated caspase-3. All these events could not be observed when CASP2L/AS cells were similarly treated with anti-Fas Abs. CASP2L/AS transfection did not inhibit the formation of the DISC and no direct interaction between procaspase-2L and either Fas or FADD or procaspase-8 was identified. Down-regulation of procaspase-2L inhibited anti-Fas Ab-mediated cleavage of c-FLIP (FLICE-inhibitory protein), a protein that interferes with the formation of a functional DISC. These results suggest that the long isoform of caspase-2 plays a role in the Fas-mediated pathway to cell death by contributing to caspase-8 activation at the DISC level.

Glutathione is implied in the control of 7-ketocholesterol-induced apoptosis, which is associated with radical oxygen species production.

In a number of experimental systems, inhibition of apoptosis by antioxidants has led to the production of radical oxygen species (ROS) in certain apoptotic forms of cell death. Since antioxidant therapies can reduce vascular dysfunctions in hypercholesterolemic patients who frequently have increased plasma levels of oxysterols constituting potent inducers of apoptosis, we speculate that oxysterol-induced apoptosis could involve oxidative stress. Here, we tested the protective effects of the aminothiols glutathione (GSH) and N-acetylcysteine (NAC), which are two potent antioxidants, on apoptosis induced by 7-ketocholesterol in U937 cells, and we present evidence indicating that oxidative processes are involved in 7-ketocholesterol-induced cell death. Thus, GSH and NAC prevented phenomenona linked to apoptosis such as reduction of cell growth, increase cellular permeability to propidium iodide, and occurrence of nuclear condensation and/or fragmentation, and they delayed internucleosomal DNA fragmentation. In addition, cell treatment with GSH impaired cytochrome c release into the cytosol and degradation of caspase-8 occurring during cell death. During 7-ketocholesterol-induced apoptosis, we also observed a rapid decrease in cellular GSH content, oxidation of polyunsaturated fatty acids, and a production of ROS by flow cytometry with the use of the dye 2', 7'-dichlorofluorescin-diacetate; both phenomena were inhibited by GSH. Prevention of cell death by GSH and NAC does not seem to be a general rule since these antioxidants impaired etoposide (but not cycloheximide) -induced apoptosis. Taken together, our data demonstrate that GSH is implied in the control of 7-ketocholesterol-induced apoptosis associated with the production of ROS.

Mitochondria-targeting drugs arsenic trioxide and lonidamine bypass the resistance of TPA-differentiated leukemic cells to apoptosis.

Exposure of U937 human leukemic cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces their differentiation into monocyte/macrophage-like cells. This terminal differentiation is associated with a resistant phenotype to apoptosis induced by the topoisomerase II inhibitor etoposide. The inhibition occurs upstream of the mitochondrial release of cytochrome c and the activation of procaspase-2, -3, -6, -7, -8, and -9. By using cell-free systems, it was demonstrated that the mitochondrial pathway to cell death that involves mitochondrial membrane depolarization, cytochrome c release and cytosolic activation of procaspases by cytochrome c/dATP remains functional in TPA-differentiated U937 cells. Accordingly, 2 drugs recently shown to target the mitochondria, namely lonidamine and arsenic trioxide, bypass the resistance of TPA-differentiated U937 cells to classical anticancer drugs. Cell death induced by the 2 compounds is associated with mitochondrial membrane depolarization, release of cytochrome c and Smac/Diablo from the mitochondria, activation of caspases, poly(ADP-ribose) polymerase cleavage and internucleosomal DNA fragmentation. Moreover, the decreased glutathione content associated with the differentiation process amplifies the ability of arsenic trioxide to activate the mitochondrial pathway to cell death. Similar results were obtained by comparing undifferentiated and TPA-differentiated human HL60 leukemic cells. These data demonstrate that mitochondria-targeting agents bypass the resistance to classical anticancer drugs induced by TPA-mediated leukemic cell differentiation. (Blood. 2001;97:3931-3940)