Small Molecule ErbB Inhibitors Decrease Proliferative Signaling and Promote Apoptosis in Philadelphia Chromosome–Positive Acute Lymphoblastic Leukemia

The presence of the Philadelphia chromosome in patients with acute lymphoblastic leukemia (Ph⁺ALL) is a negative prognostic indicator. Tyrosine kinase inhibitors (TKI) that target BCR/ABL, such as imatinib, have improved treatment of Ph⁺ALL and are generally incorporated into induction regimens. This approach has improved clinical responses, but molecular remissions are seen in less than 50% of patients leaving few treatment options in the event of relapse. Thus, identification of additional targets for therapeutic intervention has potential to improve outcomes for Ph+ALL. The human epidermal growth factor receptor 2 (ErbB2) is expressed in ∼30% of B-ALLs, and numerous small molecule inhibitors are available to prevent its activation. We analyzed a cohort of 129 ALL patient samples using reverse phase protein array (RPPA) with ErbB2 and phospho-ErbB2 antibodies and found that activity of ErbB2 was elevated in 56% of Ph⁺ALL as compared to just 4.8% of Ph⁻ALL. In two human Ph+ALL cell lines, inhibition of ErbB kinase activity with canertinib resulted in a dose-dependent decrease in the phosphorylation of an ErbB kinase signaling target p70S6-kinase T389 (by 60% in Z119 and 39% in Z181 cells at 3 µM). Downstream, phosphorylation of S6-kinase was also diminished in both cell lines in a dose-dependent manner (by 91% in both cell lines at 3 µM). Canertinib treatment increased expression of the pro-apoptotic protein Bim by as much as 144% in Z119 cells and 49% in Z181 cells, and further produced caspase-3 activation and consequent apoptotic cell death. Both canertinib and the FDA-approved ErbB1/2-directed TKI lapatinib abrogated proliferation and increased sensitivity to BCR/ABL-directed TKIs at clinically relevant doses. Our results suggest that ErbB signaling is an additional molecular target in Ph⁺ALL and encourage the development of clinical strategies combining ErbB and BCR/ABL kinase inhibitors for this subset of ALL patients.     

α-bisabolol Is an Effective Proapoptotic Agent against BCR-ABL+ Cells in Synergism with Imatinib and Nilotinib

We showed that α-bisabolol is active against primary acute leukemia cells, including BCR-ABL⁺ acute lymphoblastic leukemias (ALL). Here we studied the activity of α-bisabolol against BCR-ABL⁺ cells using 3 cell lines (K562, LAMA-84, CML-T1) and 10 primary BCR-ABL⁺ ALL samples. We found that: (a) α-bisabolol was effective in reducing BCR-ABL⁺ cell viabilty at concentrations ranging from 53 to 73 µM; (b) α-bisabolol concentrations in BCR-ABL⁺ cellular compartments were 4- to 12-fold higher than in normal cells, thus indicating a preferential intake in neoplastic cells; (c) α-bisabolol displayed a slight to strong synergism with the Tyrosine Kinase Inhibitors (TKI) imatinib and nilotinib: the combination of α-bisabolol+imatinib allowed a dose reduction of each compound up to 7.2 and 9.4-fold respectively, while the combination of α-bisabolol+nilotinib up to 6.7 and 5-fold respectively; (d) α-bisabolol-induced apoptosis was associated with loss of plasma membrane integrity, irreversible opening of mitochondrial transition pore, disruption of mitochondrial potential, inhibition of oxygen consumption and increase of intracellular reactive oxygen species. These data indicate α-bisabolol as a candidate for treatment of BCR-ABL⁺ leukemias to overcome resistance to TKI alone and to target leukemic cells through BCR-ABL-independent pathways.     

The Functional Interplay Between the t(9;22)-Associated Fusion Proteins BCR/ABL and ABL/BCR in Philadelphia Chromosome-Positive Acute Lymphatic Leukemia

The hallmark of Philadelphia chromosome positive (Ph⁺) leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph⁺ leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid leukemia (CML) and 100% of patients with Ph⁺ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96^ABL/BCR for the pathogenesis of Ph⁺ ALL. The co-expression of p96^ABL/BCR enhanced the kinase activity and as a consequence, the transformation potential of p185^BCR/ABL. Targeting p96^ABL/BCR by RNAi inhibited growth of Ph⁺ ALL cell lines and Ph⁺ ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96^ABL/BCR and p185^BCR/ABL in hematopoietic stem cells. Co-expression of p96^ABL/BCR abolished the capacity of p185^BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96^ABL/BCR for the pathogenesis of Ph⁺ ALL.     

CDKIs p18INK4c and p57Kip2 are involved in quiescence of CML leukemic stem cells after treatment with TKI

Chronic Myeloid Leukemia (CML) is sustained by a small population of cells with stem cell characteristics known as Leukemic Stem Cells that are positive to BCR-ABL fusion protein, involved with several abnormalities in cell proliferation, expansion, apoptosis and cell cycle regulation. Current treatment options for CML involve the use of Tirosine Kinase Inhibitor (Imatinib, Nilotinib and Dasatinib), that efficiently reduce proliferation proliferative cells but do not kill non proliferating CML primitive cells that remain and contributes to the persistence of the disease.

In order to understand the role of Cyclin Dependent Kinase Inhibitors in CML LSC permanence after TKI treatment, in this study we analyzed cell cycle status, the levels of several CDKIs and the subcellular localization of such molecules in different CML cell lines, as well as primary CD34⁺CD38^?lin^? LSC and HSC.

Our results demonstrate that cellular location of p18^INK4c and p57^Kip2 seems to be implicated in the antiproliferative activity of Imatinib and Dasatinib in CML cells and also suggest that the permanence of quiescent stem cells after TKI treatment could be associated with a decrease in p18^INK4c and p57^Kip2 nuclear location. The differences in p18^INK4cand p57^Kip2activities in CML and normal stem cells suggest a different cell cycle regulation and provide a platform that could be considered in the development of new therapeutic options to eliminate LSC.     

Apoptosis and neutrophils in the regulation of Ph-positive myeloid cell proliferation and differentiation ex vivo

Ex vivo proliferation and differentiation of Philadelphia chromosome-positive (Ph⁺) human myeloid cells (Ph+ cells) from chronic myeloid leukemia (CML) proceed via alternation stages of cell proliferation and neutrophil maturation. To regulate them, apoptosis is alternately blocked or induced with the help of neutrophils and expression of bcr/abl, bax, and bcl2. The regulation of apoptosis in main types of Ph⁺ cells depends on the alternation of (1) Ph⁺ cell proliferation and (2) neutrophil maturation and may follow two pathways. One consists in alternating blockages and inductions of apoptosis with initial maturation and subsequent proliferation under alternation stages as (2)-(1)-(2) and has not been described as yet. Neutrophil accumulation blocks apoptosis. As neutrophils are depleted, apoptosis is induced again. Its block accelerates proliferation with a new accumulation of neutrophils, which is followed by regular neutrophil death and a new induction of apoptosis. The way optimizes the proliferation efficiency (P/D index) with a regular alternation of maturation and proliferation, allowing the cycle of proliferation and differentiation to be completed. In another way, the alternation starts with proliferation as (1)-(2)-(1) at a lower neutrophil content) and leads to resistant decrease of the maximal apoptosis level by a factor of 3–8 as compared with (2)-(1)-(2) alternation. A stable block of apoptosis is observed in cells with prolonged stages of proliferation and maturation, leading to an accumulation of blasts and myelocytes with elevated bcr/abl expression and expression of bcl2 > bax. A stable block of apoptosis is associated with CML progression and in Ph⁺ cell lines. Cells follow the first pathway of the apoptotic regulation in chronic-phase CML. Ex vivo cultivation of Ph⁺ cells from individual CML patients was assumed to provide for a more exact diagnosis of the CML phase and optimizing the treatment.     

XBP1 promotes NRASG12D pre-B acute lymphoblastic leukaemia through IL-7 receptor signalling and provides a therapeutic vulnerability for oncogenic RAS

Activating point mutations of the RAS gene act as driver mutations for a subset of precursor-B cell acute lymphoblastic leukaemias (pre-B ALL) and represent an ambitious target for therapeutic approaches. The X box-binding protein 1 (XBP1), a key regulator of the unfolded protein response (UPR), is critical for pre-B ALL cell survival, and high expression of XBP1 confers poor prognosis in ALL patients. However, the mechanism of XBP1 activation has not yet been elucidated in RAS mutated pre-B ALL. Here, we demonstrate that XBP1 acts as a downstream linchpin of the IL-7 receptor signalling pathway and that pharmacological inhibition or genetic ablation of XBP1 selectively abrogates IL-7 receptor signalling via inhibition of its downstream effectors, JAK1 and STAT5. We show that XBP1 supports malignant cell growth of pre-B NRAS^G12D ALL cells and that genetic loss of XBP1 consequently leads to cell cycle arrest and apoptosis. Our findings reveal that active XBP1 prevents the cytotoxic effects of a dual PI3K/mTOR pathway inhibitor (BEZ235) in pre-B NRAS^G12D ALL cells. This implies targeting XBP1 in combination with BEZ235 as a promising new targeted strategy against the oncogenic RAS in NRAS^G12D-mutated pre-B ALL.     

Synergistic mitosis-arresting effects of arsenic trioxide and paclitaxel on human malignant lymphocytes

The treatment outcome of acute lymphoblastic leukemia (ALL) has improved steadily over the last 50 years. However, the cure rates are unlikely to be raised further with current therapies. Since increasing the dosage of chemotherapeutic agents could also elevate toxicity, a solution to how one could achieve maximum therapeutic effect with the minimum dosage possible is imminent. One possibility is the employment of combination drug therapies. Arsenic trioxide (ATO) is a widely used drug for acute promyelocytic leukemia (APL). Its combination with other drugs presented therapeutic activities in malignant cancers other than APL. Considering the fact that ATO induces mitotic arrest prior to apoptosis induction, we attempted to investigate the potential anti-cancer effects of ATO in combination with the microtubule-stabilizing agent, paclitaxel (PTX), using malignant lymphocytes as in vitro models. Three malignant lymphocytic cell lines and primary cells were treated with ATO and/or PTX. Using the Chou–Talalay analysis for evaluation of combined effect of ATO and PTX, we found a synergistic effect of the two drugs in the inhibition of cell growth. We also found that the combination of ATO and PTX at low concentrations synergistically induced mitotic arrest followed by apoptosis in malignant lymphocytes, which increased phosphorylated cyclin-dependent kinase 1 (Cdk1) on Thr¹⁶¹ and promoted the dysregulated activation of Cdk1. The ATO/PTX combination also significantly enhanced the activation of spindle checkpoint by inducing the formation of the inhibitory checkpoint complex BubR1/Cdc20. Our study provided the first in vitro demonstration that low concentrations of ATO and PTX synergistically induce mitotic arrest in malignant lymphocytes.     

Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome–positive acute lymphoblastic leukemia

Background aimsTo develop a treatment option for Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph⁺ALL) resistant to tyrosine kinase inhibitors (TKIs), we evaluated the anti-leukemic activity of T cells non-virally engineered to express a CD19-specific chimeric antigen receptor (CAR).MethodsA CD19.CAR gene was delivered into mononuclear cells from 10 mL of blood of healthy donors through the use of piggyBac-transposons and the 4-D Nucleofector System. Nucleofected cells were stimulated with CD3/CD28 antibodies, magnetically selected for the CD19.CAR, and cultured in interleukin-15–containing serum-free medium with autologous feeder cells for 21 days. To evaluate their cytotoxic potency, we co-cultured CAR T cells with seven Ph⁺ALL cell lines including three TKI-resistant (T315I-mutated) lines at an effector-to-target ratio of 1:5 or lower without cytokines.ResultsWe obtained ∼1.3 × 10⁸ CAR T cells (CD4⁺, 25.4%; CD8⁺, 71.3%), co-expressing CD45RA and CCR7 up to ∼80%. After 7-day co-culture, CAR T cells eradicated all tumor cells at the 1:5 and 1:10 ratios and substantially reduced tumor cell numbers at the 1:50 ratio. Kinetic analysis revealed up to 37-fold proliferation of CAR T cells during a 20-day culture period in the presence of tumor cells. On exposure to tumor cells, CAR T cells transiently and reproducibly upregulated the expression of transgene as well as tumor necrosis factor–related apoptosis-inducing ligand and interleukin-2.ConclusionsWe generated a clinically relevant number of CAR T cells from 10 mL of blood through the use of piggyBac-transposons, a 4D-Nulcleofector, and serum/xeno/tumor cell/virus-free culture system. CAR T cells exhibited marked cytotoxicity against Ph⁺ALL regardless of T315I mutation. PiggyBac-mediated CD19-specific T-cell therapy may provide an effective, inexpensive and safe option for drug-resistant Ph⁺ALL.     

Metformin Induces Apoptosis through AMPK-Dependent Inhibition of UPR Signaling in ALL Lymphoblasts

The outcome of patients with resistant phenotypes of acute lymphoblastic leukemia (ALL) or those who relapse remains poor. We investigated the mechanism of cell death induced by metformin in Bp- and T-ALL cell models and primary cells, and show that metformin effectively induces apoptosis in ALL cells. Metformin activated AMPK, down-regulated the unfolded protein response (UPR) demonstrated by significant decrease in the main UPR regulator GRP78, and led to UPR-mediated cell death via up-regulation of the ER stress/UPR cell death mediators IRE1α and CHOP. Using shRNA, we demonstrate that metformin-induced apoptosis is AMPK-dependent since AMPK knock-down rescued ALL cells, which correlated with down-regulation of IRE1α and CHOP and restoration of the UPR/GRP78 function. Additionally rapamycin, a known inhibitor of mTOR-dependent protein synthesis, rescued cells from metformin-induced apoptosis and down-regulated CHOP expression. Finally, metformin induced PIM-2 kinase activity and co-treatment of ALL cells with a PIM-1/2 kinase inhibitor plus metformin synergistically increased cell death, suggesting a buffering role for PIM-2 in metformin’s cytotoxicity. Similar synergism was seen with agents targeting Akt in combination with metformin, supporting our original postulate that AMPK and Akt exert opposite regulatory roles on UPR activity in ALL. Taken together, our data indicate that metformin induces ALL cell death by triggering ER and proteotoxic stress and simultaneously down-regulating the physiologic UPR response responsible for effectively buffering proteotoxic stress. Our findings provide evidence for a role of metformin in ALL therapy and support strategies targeting synthetic lethal interactions with Akt and PIM kinases as suitable for future consideration for clinical translation in ALL.     

Anlotinib exerts potent antileukemic activities in Ph chromosome negative and positive B-cell acute lymphoblastic leukemia via perturbation of PI3K/AKT/mTOR pathway

ObjectivesDespite advances in the development of novel targeted therapies, the need for B-ALL alternative treatments has not been met. Anlotinib could blunt the proangiogenic activity of VEGFR, PDGFR, and FGFR, and has shown strong antitumor activities across multiple tumors. However, anlotinib cytotoxicity against B-ALL has not ever been evaluated, thus prompting us to initiate this study.MethodsExpression2Kinases program was used to identify potential treatment targets. Cell viability and apoptosis were determined by CCK-8 and Annexin V/PI staining kit, respectively. qRT-PCR and Western blotting were utilized to investigate the molecular mechanisms. In vivo antileukemia activity of Anlotinib was evaluated in a Ph⁺ B-ALL patient-Derived Xenograft (PDX) model.ResultsCompared with treatment-naive B-ALL cases, RR B-ALL patients had higher activities in the VEGF/VEGFR signaling and the PI3K/AKT/mTOR pathway. Exposure of Ph⁻ and Ph⁺ B-ALL cells to anlotinib resulted in significant cell viability reduction, apoptosis enhancement, and cell cycle arrest at G2/M phase. Importantly, anlotinib treatment led to remarkably decreased leukemia burdens and extended the survival period in a Ph⁺ B-ALL PDX model. Blockade of the role of the proangiogenic mediators, comprising VEGFR2, PDGFR-beta, and FGFR3, played a critical role in the cytotoxicity of anlotinib against Ph⁻ and Ph⁺ B-ALL. Moreover, anlotinib dampened the activity of PI3K/AKT/mTOR pathway that resides in the convergence of the three mentioned proangiogenic signals.ConclusionThis work provides impressive preclinical evidence of anlotinib against Ph⁻ and Ph⁺ B-ALL and raises a rationale for future clinical evaluation of this drug in the management of Ph⁻ and Ph⁺ B-ALL.     

Coupling of the Cell Cycle and Apoptotic Machineries in Developing T Cells

Proliferation and apoptosis are diametrically opposite processes. Expression of certain genes like c-Myc, however, can induce both, pointing to a possible linkage between them. Developing CD4⁺CD8⁺ thymocytes are intrinsically sensitive to apoptosis, but the molecular basis is not known. We have found that these noncycling cells surprisingly express many cell cycle proteins. We generated transgenic mice expressing a CDK2 kinase-dead (CDK2-DN) protein in the T cell compartment. Analysis of these mice showed that the CDK2-DN protein acts as a dominant negative mutant in mature T cells as expected, but surprisingly, it acts as a dominant active protein in CD4⁺CD8⁺ thymocytes. The levels of CDK2 kinase activity, cyclin E, cyclin A, and other cell cycle proteins in transgenic CD4⁺CD8⁺ thymocytes are increased. Concurrently, caspase levels are elevated, and apoptosis is significantly enhanced in vitro and in vivo. E2F-1, the unique E2F member capable of inducing apoptosis when overexpressed, is specifically up-regulated in transgenic CD4⁺CD8⁺ thymocytes but not in other T cell populations. These results demonstrate that the cell cycle and apoptotic machineries are normally linked, and expression of cell cycle proteins in developing T cells contributes to their inherent 1sensitivity to apoptosis.     

Inhibition of Na+/K+-ATPase may be one mechanism contributing to potassium efflux and cell shrinkage in CD95-induced apoptosis

To investigate the involvement of K⁺ efflux in apoptotic cell shrinkage, we monitored efflux of the K⁺ congener,⁸⁶ Rb⁺, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in ⁸⁶Rb⁺ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed ⁸⁶Rb⁺ efflux and cell shrinkage induced by the anti- CD95 antibody. A wide range of inhibitors against most types of K⁺ channels could not inhibit CD95-mediated efflux of⁸⁶ Rb⁺, however, the uptake of⁸⁶ Rb⁺ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of⁸⁶ Rb⁺ in Jurkat cells was sensitive to ouabain (a specific Na⁺/K⁺-ATPase inhibitor), demonstrating Na⁺/K⁺-ATPase dependent K⁺ uptake. Ouabain induced significant⁸⁶ Rb⁺ efflux in untreated cells, as well as it seemed to compete with⁸⁶ Rb⁺ efflux induced by the anti-CD95 antibody, supporting a role for Na⁺/K⁺-ATPase in the CD95-mediated⁸⁶ Rb⁺ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na⁺+/K⁺-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.     

Kv 1.1 is associated with neuronal apoptosis and modulated by protein kinase C in the rat cerebellar granule cell

Previously, we reported that apoptosis of cerebellar granular neurons induced by low‐K⁺ and serum‐free (LK‐S) was associated with an increase in the A‐type K⁺ channel current (I_A), and an elevated expression of main α‐subunit of the I_A channel, which is known as Kv4.2 and Kv4.3. Here, we show, as assessed by quantitative RT‐PCR and whole‐cell recording, that besides Kv4.2 and Kv4.3, Kv1.1 is very important for I_A channel. The expression of Kv1.1 was elevated in the apoptotic neurons, while silencing Kv1.1 expression by siRNA reduced the I_A amplitude of the apoptotic neuron, and increased neuron viability. Inhibiting Kv1.1 current by dendrotoxin‐K evoked a similar effect of reduction of I_A amplitude and protection of neurons. Applying a protein kinase C (PKC) activator, phorbol ester acetate A (PMA) mimicked the LK‐S‐induced neuronal apoptotic effect, enhanced the I_A amplitude and reduced the granule cell viability. The PKC inhibitor, bisindolylmaleimide I and Gö6976 protected the cell against apoptosis induced by LK‐S. After silencing the Kv1.1 gene, the effect of PMA on the residual K⁺ current was reduced significantly. Quantitative RT‐PCR and Western immunoblot techniques revealed that LK‐S treatment and PMA increased the level of the expression of Kv1.1, in contrast, bisindolylmaleimide I inhibited Kv1.1 expression. In addition, the activation of the PKC isoform was identified in apoptotic neurons. We thus conclude that in the rat cerebellar granule cell, the I_A channel associated with apoptotic neurons is encoded mainly by the Kv1.1 gene, and that the PKC pathway promotes neuronal apoptosis by a brief modulation of the I_A amplitude and a permanent increase in the levels of expression of the Kv1.1 α‐subunit.     

Combined anticancer effects of simvastatin and arsenic trioxide on prostate cancer cell lines via downregulation of the VEGF and OPN isoforms genes

Arsenic trioxide (ATO) and statins have been demonstrated to have anti‐neoplastic properties; however, the data regarding their combination therapy is limited. Thus, we aimed to study the effects of ATO, Simvastatin and their combination in proliferation, apoptosis and pathological angiogenesis in prostate cancer cell lines. The human prostate cell lines were treated with different concentrations of Simvastatin and ATO alone and combined to find effective doses and IC50 values. In addition, the percentage of apoptotic cells was evaluated by annexin/PI staining, and mRNA expression levels of the apoptotic gene, including OPN isoforms and VEGF, were investigated using real‐time PCR. Our data displayed that Simvastatin (12 and 8 μM in PC3 and LNCaP cell lines respectively), ATO (8 and 5 μM in PC3 and LNCaP cell lines respectively), and also their combination (12 μM Simvastatin and 8 μM ATO in PC3, 8 μM Simvastatin and 5 μM ATO in LNCaP cell lines respectively) significantly increased the percentage of apoptotic cells. Also, we showed that the combination therapy by Simvastatin and ATO increased cell apoptosis and inhibited cell proliferation, providing anti‐proliferative and anti‐angiogenic properties, possibly via downregulation of the expression of VEGF and OPN genes. These results provide new perceptions regarding the anticancer roles of ATO and statins’ combination therapy in prostate cancer.     

Attenuation of apoptotic DNA fragmentation by amiloride

Amiloride is a K⁺-sparing diuretic that effectively inhibits the Na⁺/H⁺ transporter in the plasma membrane of most mammalian cells. We have examined the effects of amiloride on the progression of apoptosis in HL-60 cells induced by camptothecin (CAM), cycloheximide (CHX), and 20 Gy gamma irradiation. Spectrofluorometric measurements on cell populations showed an inhibition of Na⁺/H⁺ transporter activity and a corresponding decrease in intracellular pH following treatment with amiloride alone, or in combination with the apoptosis-inducing agents. Flow cytometric cell cycle analysis, in combination with DNA strand break analysis, indicated that amiloride diminished endonuclease-mediated degradation of nuclear chromatin 3 h following treatment with CAM or CHX, and prevented degradation for 3 h following gamma radiation treatment. Apoptosis-associated DNA degradation was significantly greater for all three agents in the absence of amiloride. Protection from radiation-induced apoptosis was transient, since apoptotic subpopulations were observed, but still at a decreased level, 5 h following irradiation. Amiloride was as effective as zinc, an inhibitor of Ca²⁺/Mg²⁺-dependent endonucleases, in reducing or delaying the onset of endonuclease activity. Data presented show that effects of amiloride on membrane Na⁺/H⁺ transporter activity and intracellular pH can potentially affect apoptotic signaling cascades, leading to a retardation in the rate of progression to an apoptotic cell death. Results also point to the involvement of intracellular pH and Ca²⁺ in the regulation of apoptotic endonuclease activity, and the need for a functional Na⁺/H⁺ exchanger for the induction of apoptosis. J. Cell. Physiol. 175:59–67, 1998. © 1998 Wiley-Liss, Inc.     

Role of Ca2+, Mg2+ and K+ ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture

We have addressed the possibility that Ca²⁺, Mg²⁺ and K⁺ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death. By removing Ca²⁺, Mg²⁺ or K⁺ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability. The differences were explained in terms of a possible reduction in their respective intracellular levels. From several lines of evidence, the deprivation of K⁺ ions was the most detrimental to cellular growth and viability and induced significant levels of early apoptotic cells. Another effect of this deprivation was to weaken the plasma membranes without causing membrane breakdown; exposure to high agitation rates confirmed fragility of the cell membranes. Removal of Mg²⁺ caused a reduction in the levels of early apoptotic cells and predisposed cells to high levels of primary necrotic death. The lower levels of apoptotic cells failed to demonstrate the classic nuclear morphology associated with apoptosis, while retaining other apoptotic features. These results highlighted the importance of utilizing several assays for the determination of apoptosis. The absence of Ca²⁺ appeared to be the mildest insult, but its deprivation did accelerate a significant decline in culture by increasing apoptotic death. Hybridoma cells overexpressing the apoptotic suppresser gene bcl-2 were protected from the predominantly necrosis inducing effects of Mg²⁺ ion deprivation and apoptosis inducing effects of Ca²⁺ ion deprivation. However, apoptosis was not as effectively suppressed in bcl-2 cells responding to incubation in K⁺ free medium. The inclusion of bcl-2 activity in the mechanisms of Ca²⁺ Mg²⁺ or K⁺ deprivation induced cell death emphasizes a close relationship between ionic dissipation and the apoptotic process.     

Proteasome inhibition activates the mitochondrial pathway of apoptosis in human CD4+ T cells

We have previously shown that inhibition of the proteolytic activity of the proteasome induces apoptosis and suppresses essential functions of activated human CD4⁺ T cells, and we report now the detailed mechanisms of apoptosis following proteasome inhibition in these cells. Here we show that proteasome inhibition by bortezomib activates the mitochondrial pathway of apoptosis in activated CD4⁺ T cells by disrupting the equilibrium of pro‐apoptotic and anti‐apoptotic proteins at the outer mitochondrial membrane (OMM) and by inducing the generation of reactive oxygen species (ROS). Proteasome inhibition leads to accumulation of pro‐apoptotic proteins PUMA, Noxa, Bim and p53 at the OMM. This event provokes mitochondrial translocation of activated Bax and Bak homodimers, which induce loss of mitochondrial membrane potential (ΔΨm). Breakdown of ΔΨm is followed by rapid release of pro‐apoptotic Smac/DIABLO and HtrA2 from mitochondria, whereas release of cytochrome c and AIF is delayed. Cytoplasmic Smac/DIABLO and HtrA2 antagonize IAP‐mediated inhibition of partially activated caspases, leading to premature activation of caspase‐3 followed by activation of caspase‐9. Our data show that proteasome inhibition triggers the mitochondrial pathway of apoptosis by activating mutually independent apoptotic pathways. These results provide novel insights into the mechanisms of apoptosis induced by proteasome inhibition in activated T cells and underscore the future use of proteasome inhibitors for immunosuppression. J. Cell. Biochem. 108: 935–946, 2009. © 2009 Wiley‐Liss, Inc.     

Intracellular cytokine profile of T cells from children with acute lymphoblastic leukemia

Purpose: During an ongoing immune response, cytokines produced by T helper types 1 (Th1) and 2 (Th2) together with T cytotoxic types 1 (Tc1) and 2 (Tc2) are critical to the effectiveness of that response. Dysregulated expansion of one or the other subset may contribute to the impaired function of the T-cell-mediated immune system in cancer patients. In the present study we have investigated whether such dysregulation might exist in children with acute lymphoblastic leukemia (ALL). Methods: We analyzed 61 blood samples from 45 children with B cell precursor ALL and 16 healthy children. Interleukin(IL)-2, IL-4, and interferon γ (IFNγ) production of their respective purified CD4⁺ and CD8⁺ T cells were assessed at the single-cell level by intracellular-cytokine-staining flow cytometry. Results: At the time of diagnosis, IL-2-producing cell populations in CD4⁺ and CD8⁺ T cells were reduced below the normal range in 31 of 44 (70.5%) and 23 of 38 (60.5%) cases respectively. Similarly, IFNγ-producing cell populations in CD4⁺ and CD8⁺ T cells decreased in 17 of 44 (38.6%) and 18 of 38 (47.4%) cases respectively. Conversely cell populations capable of IL-4 production in CD4⁺ and CD8⁺ T cell subsets were increased in 13 of 30 (43.3%) and 15 of 30 (50.0%) cases respectively. Therefore, the Th1-to-Th2 and Tc1-to-Tc2 ratios (1.6 ± 2.2 and 7.7 ± 6.7 respectively) were significantly lower in peripheral blood T cells of ALL patients (n = 30) than those (6.0 ± 2.9 and 20.1 ± 10.3 respectively) in 15 healthy controls (P < 0.0001). Although both CD45RA⁺/CD4⁺ and CD45RA⁺/CD8⁺ cells significantly increased in 43 ALL patients (P < 0.05), there existed no apparent correlation between CD45 isoform expression and cytokine (IL-2 and IFNγ) production. Interestingly, the ability to produce both IL-2 and IFNγ was recovered in 8 cases examined, after complete remission had been achieved. Conclusion: These observations suggest that, in both CD4⁺ and CD8⁺ T cells of ALL patients, there is a dysregulation in the functionality of Th1 (Tc1) and Th2 (Tc2) cells with a gross reduction of Th1 (Tc1) cell populations and an expansion in Th2 (Tc2). Received: 12 November 1999 / Accepted: 2 January 2000