p53 and Notch signaling in chronic lymphocytic leukemia: clues to identifying novel therapeutic strategies.

The p53 tumor suppressor protein has a key role in the induction of apoptosis of chronic lymphocytic leukemia (CLL) cells. Abnormalities within the p53 pathway identify a subset of patients with a poor prognosis. This review describes recent advances in understanding the mechanisms that regulate p53 levels and the role of p53 in the control of the cell cycle and of apoptosis. The classical model of p53-mediated apoptosis emphasizes the transcriptional activation of proapoptotic genes. In contrast, a novel model emphasizes p53's non-transcriptional actions as the major route of apoptosis induction, whereas its transcriptional arm predominantly upregulates antiapoptotic genes, thus providing a negative feedback mechanism that limits apoptosis. Further studies have identified the Notch pathway as a candidate p53-induced antiapoptotic mechanism. In contrast to the classical model, the novel model predicts that pharmacological inhibition of p53's transcriptional function or of the Notch signaling pathway will augment apoptosis induction by cytotoxic agents. Therapeutic strategies based on the novel model, which we review here for the first time, may significantly augment the antitumor actions of cytotoxic agents in CLL and in other malignancies.

The sesquiterpene lactone parthenolide induces selective apoptosis of B-chronic lymphocytic leukemia cells in vitro.

We have studied the in vitro actions of the sesquiterpene lactone parthenolide (PTL) on cells isolated from patients with chronic lymphocytic leukemia (CLL). Dye reduction viability assays showed that the median LD(50) for PTL was 6.2 muM (n=78). Fifteen of these isolates were relatively resistant to the conventional agent chlorambucil but retained sensitivity to PTL. Brief exposures to PTL (1-3 h) were sufficient to induce caspase activation and commitment to cell death. Chronic lymphocytic leukemia cells were more sensitive towards PTL than were normal T lymphocytes or CD34(+) haematopoietic progenitor cells. The mechanism of cell killing was via PTL-induced generation of reactive oxygen species, resulting in turn in a proapoptotic Bax conformational change, release of mitochondrial cytochrome c and caspase activation. Parthenolide also decreased nuclear levels of the antiapoptotic transcription factor nuclear factor-kappa B and diminished phosphorylation of its negative regulator IkappaB. Killing of CLL cells by PTL was apparently independent of p53 induction. This is the first report showing the relative selectivity of PTL towards CLL cells. The data here warrant further investigation of this class of natural product as potential therapeutic agents for CLL.

CCK2 gastrin receptor as a potential target for therapy in leukaemia cell lines.

The gastrin CCK2 pathway has been implicated in the development of various cancers including leukaemia. An autocrine or intracrine pathway may exist in the leukaemia cell that is involved in stimulating proliferation. We tested four leukaemia cell lines, KU812, ML-1, MOLT-4 and U937 for the existence of the CCK2 receptor and gastrin precursor protein using immunoblotting. We also assessed the effect of CCK2 antagonist PD 135 and both gastrin 17 and glycine-extended gastrin on the proliferation of the cell lines. We found immunoreactive CCK2 and gastrin precursors present in all 4 cell lines. We also observed a stimulatory effect on proliferation by gastrin and glycine-extended gastrin on 2 and 3 of the cell lines respectively and an inhibitory effect of PD 135 on all 4 cell lines. These results demonstrate that the gastrin-gastrin receptor axis is a potential target for new therapeutic strategies.

Caspase 8 activation independent of Fas (CD95/APO-1) signaling may mediate killing of B-chronic lymphocytic leukemia cells by cytotoxic drugs or gamma radiation.

Ligation of the cell-surface Fas molecule by its ligand (Fas-L) or agonistic anti-Fas monoclonal antibodies results in the cleavage and activation of the cysteine protease procaspase 8 followed by the activation of procaspase 3 and by apoptosis. In some leukemia cell lines, cytotoxic drugs induce expression of Fas-L, which may contribute to cell killing through the ligation of Fas. The involvement of Fas, Fas-L, and caspase 8 was studied in the killing of B-cell chronic lymphocytic leukemia (B-CLL) cells by chlorambucil, fludarabine, or gamma radiation. Spontaneous apoptosis was observed at 24-hour incubation, with additional apoptosis induced by each of the cytotoxic treatments. Although Fas mRNA expression was elevated after exposure to chlorambucil, fludarabine, or gamma radiation, Fas protein levels only increased after irradiation. Therefore, Fas expression may be regulated by multiple mechanisms that allow the translation of Fas mRNA only in response to restricted cytotoxic stimuli. None of the cytotoxic stimuli studied here induced Fas-L expression. An agonistic anti-Fas monoclonal antibody (CH-11) did not significantly augment apoptosis induction by any of the death stimuli. A Fas-blocking antibody (ZB4) did not inhibit spontaneous, chlorambucil-, fludarabine-, or radiation-induced apoptosis. However, procaspase 8 processing was induced by all cytotoxic stimuli. These data suggest that the Fas/Fas-L signaling system does not play a major role in the induction of apoptosis in B-CLL cells treated with cytotoxic drugs or radiation. However, Fas-independent activation of caspase 8 may play a crucial role in the regulation of apoptosis in these cells.

Autologous plasma activates Akt/protein kinase B and enhances basal survival and resistance to DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia cells.

We have studied the actions of autologous plasma on both basal and DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia (B-CLL) cells. Apoptosis was quantified using morphological criteria and Western blot analysis for the apoptosis-specific p85 fragment of poly(ADP ribose) polymerase. Cell viability was estimated using the methyl thiazol tetrazolium bromide dye reduction assay. Plasma cultures showed lower rates of basal apoptosis as well as a decreased cytotoxic response to chlorambucil and gamma-radiation compared with cultures in fetal calf serum. Experiments using neutralizing antibodies suggested that the protective actions of plasma could not be accounted for by interleukin 4, the interferons alpha or gamma or stromal cell-derived factor 1, each of which have been shown to protect B-CLL cells from apoptosis in vitro. Plasma addition to B-CLL cells resulted in rapid activation of the Akt protein kinase, a key signalling enzyme that has been implicated in anti-apoptotic signalling. LY294002, an inhibitor of phosphatidylinositol 3'-kinase, blocked Akt activation by plasma. To the best of our knowledge, this is the first report to show that factors present in plasma promote basal survival of B-CLL cells and resistance to cytotoxic drugs via stimulation of the Akt cytoprotective-signalling pathway. Pharmacological blockade of this pathway may have potential in the development of novel therapeutic strategies for B-CLL treatment.

Antioxidants enhance the susceptibility of colon carcinoma cells to 5-fluorouracil by augmenting the induction of the bax protein.

5 Fluorouracil (5 FU), the most effective systemic chemotherapeutic agent in the management of advanced colorectal carcinoma acts by inducing apoptosis. Response rates, approximately 20% is improved by folinic acid. This study investigates similar modulation of 5 FU-induced apoptosis by oxidant quenching. A five-fold reduction of intracellular oxidant levels by antioxidants N-acetylcysteine and vitamin E did not induce apoptosis, it however augmented pro-apoptotic bax protein expression, and apoptotic response to a non-toxic dose of 5 FU in the colorectal cancer cell lines colo 201 and colo 205. This suggests that reduction of intracellular levels of reactive oxygen species enhance susceptibility to 5 FU (apoptotic stimuli) by augmentation of bax expression.

Killing of T lymphocytes by synthetic ceramide is by a nonapoptotic mechanism and is abrogated following mitogenic activation.

Ceramide induces apoptosis in leukemia cell lines and has been proposed as a potential therapeutic agent in malignancies refractory to conventional treatment. Here we show that synthetic N-acetyl-d-erythro-sphingosine (C2 ceramide) kills normal human T lymphocytes by a caspase-independent nonapoptotic mechanism. By contrast, T cells were induced to caspase-dependent apoptosis by okadaic acid. Furthermore, C2 ceramide treatment of the Jurkat leukemia cell line induced killing by apoptosis. Activation of T lymphocytes by phytohemagglutinin abrogated killing by C2 ceramide. The data here suggest that ceramide triggers caspase-dependent apoptosis in leukemia cells lines, but activates caspase-independent nonapoptotic killing of resting T lymphocytes which is abrogated following mitogenic activation.

Ceramide-induced killing of normal and malignant human lymphocytes is by a non-apoptotic mechanism.

Synthetic ceramides induce apoptotic death of Jurkat and HL60 leukaemia cell lines. By contrast we show here that ceramide induces non-apoptotic killing of malignant cells from patients with B-chronic lymphocytic leukaemia (B-CLL) and of normal B lymphocytes. The protein phosphatase inhibitor okadaic acid readily induces apoptosis of B-CLL cells, indicating that this death pathway is fully functional in these cells. The ability of ceramide to activate the apoptotic protease caspase 3 in HL60 cells but not in B-CLL cells, as well as the lack of correlation of ceramide-mediated killing of different B-CLL isolates with expression of the apoptosis-regulating proteins bcl-2 and bax reinforce the conclusion that ceramide killing of B-CLL cells is by a non-apoptotic mechanism. Fludarabine treatment or gamma-irradiation of B-CLL cells resulted in ceramide elevation and in killing by both apoptotic and non-apoptotic mechanisms, suggesting that a ceramide-triggered non-apoptotic mechanism may play a role in the killing of these cells. Therefore, the results here show that ceramide can induce either apoptotic or non-apoptotic death, depending on the cellular context. The inability of synthetic dihydroceramide to kill B-CLL cells or normal B lymphocytes suggests that non-apoptotic killing by ceramide is via interaction with a specific, but unidentified, cellular target.

Integrin signalling defects in T-lymphocytes in systemic lupus erythematosus.

OBJECTIVE: To establish the relationship between T cell responses to integrin coreceptor stimulation and B cell hyperreactivity as measured by pathologic autoantibody production. METHODS: Peripheral blood mononuclear cells from 42 patients with SLE according to the American Rheumatism Association criteria were examined for their ability to adhere to plate-immobilised fibronectin. Co-stimulation assays were performed on the same cells using anti-CD3 antibody alone or co-immobilised with an anti-beta1-integrin antibody. Proliferative responses were measured by 3[H]thymidine pulsing on day 3 and activation was determined using a commercial protein kinase C assay, the protocol being established by our group in association with Promega. Beta-integrin expression was established by FACS analysis. RESULTS: An impaired PKC response to integrin-mediated activation was found in T-lymphocytes from 6/21 (29%) SLE patients, which correlated significantly with an absence of anti-dsDNA antibody in patient sera, irrespective of prednisolone treatment. Integrin co-stimulation of TcR/CD3-induced proliferation and T cell adhesion to fibronectin were also impaired among 5/21 (24%) and 6/15 (40%) patients studied, respectively. CONCLUSION: We hypothesise that the integrity of beta1-integrin signalling pathways may influence pathological antibody production in SLE by affecting T-lymphocyte activation and interactions between T- and B-lymphocytes.

Monocytes stimulate expression of the Bcl-2 family member, A1, in endothelial cells and confer protection against apoptosis.

We have investigated the molecular mechanisms underlying the ability of peripheral blood monocytes to block apoptosis induction in endothelial cells. Monocytes stimulated the expression of the bcl-2 homologue A1 in serum-starved endothelial cells after 6 h of coincubation, with elevated A1 levels persisting for up to 21 h. IL-1 and TNF also stimulated A1 expression at 6 h, but A1 transcript levels fell by 21 h. Direct cellular contact with monocytes was required for stimulation of A1 mRNA in endothelial cells. Stimulation of endothelial cell A1 mRNA by monocytes was not inhibited by anti-beta2 integrin Abs, but anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) mAb reduced A1 transcript levels at 21 h. Studies employing either TNF on its own, or anti-TNF in endothelium/monocyte cocultures showed that TNF plays a role in the early (6-h) stimulation of A1, but is less important for the sustained elevation of A1 levels at 21 h. Serum-starved endothelial cells demonstrated increased survival and decreased apoptosis after coculture with monocytes. IL-10 reduced A1 mRNA expression in, as well as survival of, endothelial cells that were cocultured with monocytes. In comparison with A1, Bcl-2 was expressed at low levels and was up-regulated by monocytes only at 21 h, while neither Bax nor Bcl-xL levels were altered by monocytes. The interaction of monocytes with endothelium during the course of an inflammatory reaction may provide survival signals to endothelial cells.

Activation-associated necrosis in human immunodeficiency virus infection.

Mitogenic stimulation of lymphocytes from persons infected with human immunodeficiency virus (HIV) resulted in massive cell death. In addition to early apoptosis, a second wave of cell death occurred 48-72 h after stimulation. At that time, the cells were enlarged, leaked content, and had plasma membrane damage-all indicative of necrosis. Furthermore, DNA fragmentation as determined by TUNEL assay was virtually absent. This activation-associated necrosis could not be prevented by interfering with CD95/CD95-ligand interactions or by blocking caspase activity and was unaffected by neutralizing antibodies to tumor necrosis factor-alpha or interferon-gamma. Necrosis was also induced by activation of normal lymphocytes in the presence of ribavirin, which inhibits the de novo pathway of nucleotide synthesis. Lymphocytes from HIV-infected persons are defective in their ability to synthesize nucleotides via this pathway, indicating one possible mechanism for the activation-associated necrosis seen in HIV infection.

Okadaic acid-induced apoptosis of HL60 leukemia cells is preceded by destabilization of bcl-2 mRNA and downregulation of bcl-2 protein.

We have studied the actions of the protein phosphatase inhibitor okadaic acid (OA) on the expression of bcl-2 in HL60 human leukemia cells. OA induced downregulation of bcl-2 mRNA and protein prior to the induction of apoptosis. Downregulation of bcl-2 mRNA levels did not result from actions of OA on the bcl-2 upstream negative response element. Nuclear run-off analyses confirmed that OA did not affect bcl-2 gene transcription. However, OA caused a rapid increase in the rate of degradation of bcl-2 mRNA. Therefore, OA induces down-regulation of bcl-2 expression via destabilization of its transcript. The constitutive action of an OA-sensitive protein phosphatase may therefore maintain HL60 cell survival by blocking bcl-2 mRNA degradation.

Herbimycin A accelerates the induction of apoptosis following etoposide treatment or gamma-irradiation of bcr/abl-positive leukaemia cells.

Philadelphia chromosome (Ph)-positive leukaemia cells express the chimeric bcr/abl oncoprotein, whose deregulated protein tyrosine kinase (PTK) activity antagonizes the induction of apoptosis by DNA damaging agents. Treatment of Ph-positive K562, TOM 1 and KCL-22 cells with etoposide for 2d induced cytosolic vacuolation, but not nuclear condensation or DNA fragmentation. The bcr/abl kinase-selective inhibitor herbimycin A increased the induction of nuclear apoptosis by etoposide or gamma-radiation. The concentration of herbimycin required to synergize with etoposide was similar to that required to decrease the level of tyrosine phosphorylated proteins or of the protein tyrosine kinase activity of anti-abl immune complexes in K562 cells. The ability of herbimycin A to sensitize K562, TOM 1 or KCL-22 cells to apoptosis induction correlated with its ability to decrease the cellular content of phosphotyrosyl proteins in these Philadelphia-positive lines. Enhancement of nuclear apoptosis by herbimycin was not attributable to downregulation of the bcl-2 or bcl-XL anti-apoptotic proteins. In contrast, herbimycin protected Philadelphia-negative HL60 cells from apoptosis induction by etoposide and did not affect killing of NC37 and CEM cells. The data suggest that the induction of apoptosis is blocked in cells expressing the bcr/abl oncoprotein and that herbimycin A increases induction of programmed cell death following DNA damage. Selective PTK inhibitors may therefore be of value in securing the genetic death of Ph-positive leukaemia cells.

Signal Transduction by the Philadelphia Chromosome-encoded BCR/ABL Oncoproteins: Therapeutic Implications for Chronic Myeloid Leukemia and Philadelphia-positive Acute Lymphoblastic Leukemia.

The Philadelphia chromosomes characteristic of chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (ALL) encode chimeric protein tyrosine kinases (PTKs) derived by fusion of the normal BCR and ABL genes. The oncogenic properties of these BCR/ABL oncoproteins are dependent on their elevated PTK activity and on their ability to interact with multiple signal transduction systems. Here we summarize some of the key pathways which are activated by normal receptors with PTK activity and which modulate cell proliferation and survival. Next, we review some of the biochemical pathways initiated by BCR/ABL oncoproteins and discuss their possible relevance to the leukemic phenotype. We finally review experimental approaches designed to suppress signalling by BCR/ABL oncoproteins and discuss their potential therapeutic applications.

The integrin-triggered rescue of T lymphocyte apoptosis is blocked in HIV-1-infected individuals.

HIV infection is associated with a disease status-dependent impairment of Ag-specific T cell responses, resulting in anergy or unchecked apoptotic cell death. beta1 integrins play an important role in the induction of T lymphocyte responses to antigenic challenge by providing a T cell costimulatory signal, and have been shown to rescue various cell types from undergoing apoptosis. We examined the integrin-triggered cell survival signal and associated pathways in CD3+ T cells derived from 69 HIV-1-infected individuals in comparison with healthy controls. We found beta1 integrin-mediated costimulation of TCR-induced T cell proliferation and protection from aberrant cell death to be absent in the majority of patients with AIDS, but intact in asymptomatic, infected individuals. The lack of integrin-mediated rescue may be partly due to an early impairment of TCR/integrin-costimulated secretion of IFN-gamma, a type 1 lymphokine that protects against TCR-induced apoptosis of T cells from HIV-seropositive donors, but not loss of integrin expression. The mechanism of integrin hyporesponsiveness appeared to correlate with a failure of the integrin-generated signal to induce pp125FAK mRNA and protein expression. Protein kinase C activation in CD3+ T cells following integrin stimulation was also impaired in HIV-infected individuals, mostly among the symptomatic/AIDS patients. Protein kinase C inactivation in T cells was shown to have a destabilizing effect in vitro on pp125FAK mRNA that contains an AUUUA motif in the 3'-untranslated region, a consensus sequence for the AU-rich elements responsible for mRNA destabilization. These aberrant changes in pp125FAK expression may have direct significance to the overall immunopathogenesis during infection with HIV-1.

Co-ordinated downregulation of bcl-2 and bax expression during granulocytic and macrophage-like differentiation of the HL60 promyelocytic leukaemia cell line.

The bcl-2 protein suppresses apoptosis and the bax protein opposes the cytoprotective effect of bcl-2. A decrease in bcl-2 levels has been implicated in the induction of apoptosis during the terminal differentiation of HL60 myeloid leukaemia cells. We show here that bax protein also declined with a time course similar to the downregulation of bcl-2 following treatment of HL60 with phorbol myristate acetate (PMA), dimethyl sulphoxide (DMSO) or retinoic acid (RA). Decreased bcl-2 protein expression in induced cells was associated with down-regulation of its mRNA. By contrast, the decrease in bax occurred by a post-transcriptional mechanism. Co-ordinate downregulation of bcl-2 and bax proteins may fine-tune the induction of apoptosis during cellular differentiation.

Interleukin-2 receptor common gamma-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression.

Cytokine deprivation from activated T cells leads to apoptosis associated with down-regulation of the bcl-2 gene product. It is not clear, however, how cytokines other than interleukin-2 (IL-2) may affect this process and regulate the involvement of other apoptosis-modulating genes. We show that a group of cytokines including IL-2 (IL-2R gamma), prevent the apoptosis of IL-2-deprived activated T cells. This rescue involves the induction of the anti-apoptosis genes bcl-2 and bcl-xL), but causes little change in expression of bax and bcl-xS, which promote apoptosis. Furthermore, the prevention of apoptosis and induction of proliferation by the common gamma chain cytokines can be dissociated. Thus, when proliferation is blocked, the common gamma chain cytokines still induce up-regulation of bcl-2 relative to bax and retard apoptosis. These cytokines can thus regulate the persistence or removal of effector T cells by coordinating the balance between genes which promote and those which inhibit apoptosis, events which are probably mediated at least in part by signals through the common gamma chain. These data also implicate inappropriate T cell apoptosis resulting from a dysfunctional common gamma-chain as part of the pathophysiological defect in patients with X-linked severe-combined immunodeficiency (SCID).