Interleukin-3 Deprivation Research Articles

Treg sensitivity to FasL and relative IL-2 deprivation drive idiopathic aplastic anemia immune dysfunction

AbstractIdiopathic aplastic anemia (AA) has 2 key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T-cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predicts response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified 2 mechanisms that lead to skewed Treg composition in AA: first, FasL-mediated apoptosis on ligand interaction; and, second, relative interleukin-2 (IL-2) deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed a high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T-cell–mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy.

Flow Cytometry Analysis of Anastasis using CellTrace Far Red (CTFR) cell tracker in Cultured Mammalian T Lymphocytes

Anastasis is a cellular recovery process that rescues apoptotic cells from the brink of death. Our lab has developed a model for studying anastasis using cultured mouse HT2 lymphocytes dependent on the cytokine interleukin 2 (IL2). Within 24 hours of IL2 deprivation, HT2 cells display characteristic changes underlying apoptosis, including cellular condensation, membrane blebbing and fragmentation into apoptotic bodies, phosphatidylserine externalization, caspase 3 activation as well as G1 cell cycle arrest. HT2 cells can subsequently be rescued by adding IL2 back into the culture medium. In this study, we have used quantitative flow cytometry in conjunction with CellTrace Far Red (CTFR) and SYTOX Green (live/dead indicator) to track the kinetics of anastasis and re‐entry of viable apoptotic cells into the cell cycle. HT2 lymphocytes continuously maintained in IL2 comprised a homogeneous, viable population of CTFR+ cells which decreased in fluorescence intensity with each subsequent cell cycle for up to 96 hours post‐CTFR labelling. To investigate apoptosis reversal, HT2 cells were labeled with CTFR, deprived of IL2 for 24 hours and then rescued with IL2 re‐addition for periods of 24, 48, and 72 hours. Our findings indicate that viable, CTFR+ HT2 cells gradually resumed cell cycle entry over the 72‐hour rescue period. As caspase activation represents a cellular marker of early‐mid stage apoptotic cells, we also investigated caspase 3/7 activity in viable CTFR+ cells using the Nucview‐488 fluorescent caspase 3 substrate. Three distinct populations of caspase‐high, medium and low, viable CTFR+ HT2 cells were observed at 24 hours following IL2 deprivation. IL2‐rescued cells subsequently segregated into caspase‐high and low populations, ultimately merging into a single, caspase‐low population by 72 hours post‐rescue. Collectively, our findings suggest that CTFR tracking can be used successfully as a tool to follow the kinetics of apoptosis reversal in IL2‐dependent mouse HT2 lymphocytes, and is a promising model for the study of the mechanistic basis of anastasis.Support or Funding InformationUnion College Faculty and Student Research Grants

IL-2 Therapy Diminishes Renal Inflammation and the Activity of Kidney-Infiltrating CD4+ T Cells in Murine Lupus Nephritis.

An acquired deficiency of interleukin-2 (IL-2) and related disturbances in regulatory T cell (Treg) homeostasis play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Low-dose IL-2 therapy was shown to restore Treg homeostasis in patients with active SLE and its clinical efficacy is currently evaluated in clinical trials. Lupus nephritis (LN), a challenging organ manifestation in SLE, is characterized by the infiltration of pathogenic CD4+ T cells into the inflamed kidney. However, the role of the Treg-IL-2 axis in the pathogenesis of LN and the mode of action of IL-2 therapy in the inflamed kidneys are still poorly understood. Using the (NZB × NZW) F1 mouse model of SLE we studied whether intrarenal Treg are affected by a shortage of IL-2 in comparison with lymphatic organs and whether and how intrarenal T cells and renal inflammation can be influenced by IL-2 therapy. We found that intrarenal Treg show phenotypic signs that are reminiscent of IL-2 deprivation in parallel to a progressive hyperactivity of intrarenal conventional CD4+ T cells (Tcon). Short-term IL-2 treatment of mice with active LN induced an expansion the intrarenal Treg population whereas long-term IL-2 treatment reduced the activity and proliferation of intrarenal Tcon, which was accompanied by a clinical and histological amelioration of LN. The association of these immune pathologies with IL-2 deficiency and their reversibility by IL-2 therapy provides important rationales for an IL-2-based immunotherapy of LN.

Flow Cytometry Analysis of Anastasis in Cultured Mammalian Lymphocytes

Apoptosis is a physiological form of programmed cell death characterized by phosphatidylserine externalization, caspase activation, and genomic DNA fragmentation. Recent studies have shown that apoptosis may be reversed even in its late stages, calling the previous notion of a ‘point of no return’ into question. This recovery process has been termed anastasis (‘rising to life’), but its underlying mechanisms are not well understood. Our lab has developed a model of studying anastasis using cultured mouse HT‐2 lymphocytes dependent on the cytokine interleukin 2 (IL2). Our previous findings have shown that HT‐2 cells accumulate in the G1 stage of the cell cycle following 24 hours of IL2 deprivation and can be rescued by adding IL2 back into the culture medium. In this study, we have used quantitative flow cytometry to investigate which apoptotic events are congruent with anastasis. HT2 cells were deprived of IL2 for 24 hours and then rescued for periods of 0, 24, 48, and 72 hours. CD25 surface expression (high‐affinity IL2 receptor) and phosphatidylserine externalization (annexin V staining) were subsequently monitored prior to and following recovery, as markers of T cell activation and early stage apoptosis, respectively. Control HT2 lymphocytes continuously maintained in IL2 comprised a single viable population of CD25HIGHAnnexin−cells. Cells deprived of IL2 for 24 hours were observed to fall into two populations: CD25LOWAnnexin+early apoptotic and CD25LOWAnnexinnon‐apoptotic cells. In both populations, CD25 expression was reduced approximately 10‐fold when compared to IL2‐maintained controls. During anastasis, three distinct viable cell populations emerged: 1) CD25LOWAnnexin+cells that did not progress into the later stages of apoptosis, 2) CD25LOWAnnexin−non‐apoptotic cells, and 3) CD25HIGHAnnexin−non‐apoptotic cells. With advancing recovery time, viable CD25LOWpopulations decreased as CD25HIGHcells increased. By 72 hours post‐recovery, the majority of cells were actively cycling, annexin negative cells. These findings suggest that IL2‐dependent mouse HT‐2 lymphocytes may be a promising model to study the mechanisms of anastasis.Support or Funding InformationUnion College Faculty and Student Research GrantsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction of Cell Death in Growing Human T-Cells and Cell Survival in Resting Cells in Response to the Human T-Cell Leukemia Virus Type 1 Tax.

Tax1 encoded by the human T-cell leukemia virus type 1 (HTLV-1) has been believed to dysregulate the expression of cellular genes involved in cell survival and mortality, leading to the development of adult T-cell leukemia (ATL). The function of Tax1 in ATL development however is still controversial, primarily because Tax1 induces cell cycle progression and apoptosis. To systemically understand cell growth phase-dependent induction of cell survival or cell death by Tax1, we established a single experimental system using an interleukin 2 (IL-2)-dependent human T-cell line Kit 225 that can be forced into resting phase by IL-2 deprivation. Introduction of Tax1 and HTLV-2 Tax (Tax2B) decreased mitochondrial activity alongside apoptosis in growing cells but not in resting cells. Cell cycle profile analysis indicated that Tax1 and Tax2B were likely to perturb the S phase in growing cells. Studies with Tax1 mutants and siRNA for NF-κB/RelA revealed that Tax1-mediated cell growth inhibition and apoptosis in growing Kit 225 cells depend on RelA. Interestingly, inactivation of the non-canonical NF-κB and p38 MAPK pathways relieved Tax1-mediated apoptosis, suggesting that the Tax1-NF-κB-p38 MAPK axis may be associated with apoptosis in growing cells. Inflammatory mediators such as CCL3 and CCL4, which are involved in oncogene-induced senescence (OIS), were induced by Tax1 and Tax2B in growing cells. In contrast, RelA silencing in resting cells reduced mitochondrial activity, indicating that NF-κB/RelA is also critical for Tax1-mediated cell survival. These findings suggest that Tax1-mediated cell survival and death depend on the cell growth phase. Both effects of Tax1 may be implicated in the long latency of HTLV-1 infection.

8: Heterodimeric IL-15 promotes tumor control through the regulation of the balance of effector and regulatory cells via an IL-2 deprivation mechanism

Interleukin-15 (IL-15) is a common γ -chain cytokine that plays a significant role in the activation and proliferation of T and NK cells and holds great potential in fighting infection and cancer. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the soluble or cell-associated IL-15 receptor alpha chain that are together termed heterodimeric IL-15 (hetIL-15). HetIL-15 produced from engineered human cell lines showed favorable pharmacokinetics and bioactivity compared to monomeric IL-15 in both mice and macaques. We investigated the anti-tumor efficacy of hetIL-15, using the MC38 colon carcinoma mouse model. hetIL-15 resulted in a significant delay in tumor growth, when administered intraperitoneally every 2 days for 2 weeks. In addition, intratumoral delivery of hetIL-15 induced regression of established tumors and cured 50% of mice. Tumor-free mice were resistant to tumor challenge, demonstrating the development of specific anti-tumor immune responses. We further dissected the mechanism leading to tumor control in hetIL-15-treated mice, discovering a new interplay between IL-15 and the cognate cytokine interleukin-2 (IL-2). Repeated injections of hetIL-15 resulted in an increased CD8 + /Treg cells ratio in spleen, lymph nodes and tumor microenvironment. Interestingly, hetIL-15 induced a transient accumulation of CD8 + T cells expressing the high affinity IL-2 receptor CD25, that compete with CD25 + Treg for the available IL-2. In addition, IL-15 decreased the frequency of IL-2-producing CD4 + T cells, while increasing their ability to secrete IFN γ . These data suggest that hetIL-15 treatment limits Treg fitness through a concerted action of both CD8 + and CD4 + T cells, that results in an IL-2 deprived environment. Preclinical cancer studies support the use of hetIL-15 in tumor immunotherapy approaches to promote the development of anti-tumor responses by favoring effector over regulatory cells.

Interleukin-21 Is a Critical Regulator of CD4 and CD8 T Cell Survival during Priming under Interleukin-2 Deprivation Conditions

Optimal T cell activation and expansion require binding of the common gamma-chain (γc) cytokine Interleukin-2 (IL-2) to its cognate receptor that in turn engages a γc/Janus tyrosine kinase (Jak)3 signaling pathway. Because of its restricted expression by antigen-activated T cells and its obligatory role in promoting their survival and proliferation, IL-2 has been considered as a selective therapeutic target for preventing T cell mediated diseases. However, in order to further explore IL-2 targeted therapy, it is critical to precisely understand its role during early events of T cell activation. In this study, we delineate the role of IL-2 and other γc cytokines in promoting the survival of CD4 and CD8 T cells during early phases of priming. Under IL-2 inhibitory conditions (by neutralizing anti-IL-2 mAbs), the survival of activated CD8+ T cells was reduced, whereas CD4+ T cells remained much more resistant. These results correlated with reduced Bcl-2 expression, and mitochondrial membrane potential in CD8+ T cells in comparison to CD4+ T cells. However, using transwell co-culture assays we have found that CD4+ T cells could rescue the survival of CD8+ T cells even under IL-2 deprived conditions via secretion of soluble factors. A cytokine screen performed on CD8+ T cells cultured alone revealed that IL-21, another γc cytokine, was capable of rescuing their survival under IL-2 deprivation. Indeed, blocking the IL-21 signaling pathway along with IL-2 neutralization resulted in significantly reduced survival of both CD4+ and CD8+ T cells. Taken together, we have shown that under IL-2 deprivation conditions, IL-21 may act as the major survival factor promoting T cell immune responses. Thus, investigation of IL-2 targeted therapies may need to be revisited to consider blockade of the IL-21 signaling pathways as an adjunct to provide more effective control of T cell immune responses.

HTLV-1 Tax-activated NF-κB is involved in both cell growth and death

HTLV-1 Tax (Tax1) dysregulates the expression of cellular genes through transcriptional factors such as NF-kB, CREB and AP-1, leading to cell proliferation and immortalization. Conversely, Tax1 is reported to induce growth arrest and apoptosis. The molecular mechanism underlying growth arrest and cell death by Tax1 remains to be elucidated. The interleukin 2 (IL-2)-dependent human cell line Kit 225, in which the resting phase of cell growth is induced by deprivation of IL-2, was used to examine effects of Tax1 in growing and resting Kit 225 cells. Introduction of Tax1 reduced cell growth in growing cells, while Tax1 enhanced cell activity in resting cells. Tax1 activated the cell cycle transcription factor E2F and also increased the expression of cell cycle regulatory genes such as CDK2, CDK4 and cyclin D2 in resting cells. Tax1-induced reduction of cell growth was associated with apoptosis. No reduction in cell growth was observed in growing cells after introduction of Tax2 coded by HTLV-2, which has not been shown to link to malignant diseases. Tax1-induced cell growth reduction and apoptosis were demolished by siRNA against the NF-kB subunit RelA. In contrast, resting Kit 225 cells reduced cell activity by decreasing RelA upon Tax1 introduction. These results indicate that Tax1-activated RelA acts on both cell growth and cell death in a cell cycle-dependent manner. Functional differences between Tax1 and Tax2 may influence HTLV-1 pathogenesis.

MicroRNA-125b transforms myeloid cell lines by repressing multiple mRNA

We previously described a t(2;11)(p21;q23) chromosomal translocation found in patients with myelodysplasia or acute myeloid leukemia that leads to over-expression of the microRNA miR-125b, and we showed that transplantation of mice with murine stem/progenitor cells overexpressing miR-125b is able to induce leukemia. In this study, we investigated the mechanism of myeloid transformation by miR-125b. To investigate the consequences of miR-125b over-expression on myeloid differentiation, apoptosis and proliferation, we used the NB4 and HL60 human promyelocytic cell lines and the 32Dclone3 murine promyelocytic cell line. To test whether miR-125b is able to transform myeloid cells, we used the non-tumorigenic and interleukin-3-dependent 32Dclone3 cell line over-expressing miR-125b, in xenograft experiments in nude mice and in conditions of interleukin-3 deprivation. To identify new miR-125b targets, we compared, by RNA-sequencing, the transcriptome of cell lines that do or do not over-express miR-125b. We showed that miR-125b over-expression blocks apoptosis and myeloid differentiation and enhances proliferation in both species. More importantly, we demonstrated that miR-125b is able to transform the 32Dclone3 cell line by conferring growth independence from interleukin-3; xenograft experiments showed that these cells form tumors in nude mice. Using RNA-sequencing and quantitative real-time polymerase chain reaction experiments, we identified multiple miR-125b targets. We demonstrated that ABTB1, an anti-proliferative factor, is a new direct target of miR-125b and we confirmed that CBFB, a transcription factor involved in hematopoiesis, is also targeted by miR-125b. MiR-125b controls apoptosis by down-regulating genes involved in the p53 pathway including BAK1 and TP53INP1. This study demonstrates that in a myeloid context, miR-125b is an oncomiR able to transform cell lines. miR-125b blocks myeloid differentiation in part by targeting CBFB, blocks apoptosis through down-regulation of multiple genes involved in the p53 pathway, and confers a proliferative advantage to human and mouse myeloid cell lines in part by targeting ABTB1.

A Mammalian Siderophore Synthesized by an Enzyme with a Bacterial Homolog Involved in Enterobactin Production

Intracellular iron homeostasis is critical for survival and proliferation. Lipocalin 24p3 is an iron-trafficking protein that binds iron through association with a bacterial siderophore, such as enterobactin, or a postulated mammalian siderophore. Here, we show that the iron-binding moiety of the 24p3-associated mammalian siderophore is 2,5-dihydroxybenzoic acid (2,5-DHBA), which is similar to 2,3-DHBA, the iron-binding component of enterobactin. We find that the murine enzyme responsible for 2,5-DHBA synthesis, BDH2, is the homolog of bacterial EntA, which catalyzes 2,3-DHBA production during enterobactin biosynthesis. RNA interference-mediated knockdown of BDH2 results in siderophore depletion. Mammalian cells lacking the siderophore accumulate abnormally high amounts of cytoplasmic iron, resulting in elevated levels of reactive oxygen species, whereas the mitochondria are iron deficient. Siderophore-depleted mammalian cells and zebrafish embryos fail to synthesize heme, an iron-dependent mitochondrial process. Our results reveal features of intracellular iron homeostasis that are conserved from bacteria through humans.

Myeloid progenitor cells lacking p53 exhibit delayed up-regulation of Puma and prolonged survival after cytokine deprivation

AbstractLoss of p53-dependent apoptosis contributes to the development of hematologic malignancies and failure to respond to treatment. Proapoptotic Bcl-2 family member Puma is essential for apoptosis in HoxB8-immortalized interleukin-3 (IL-3)–dependent myeloid cell lines (FDM cells) provoked by IL-3 deprivation. p53 and FoxO3a can transcriptionally regulate Puma. To investigate which transcriptional regulator is responsible for IL-3 deprivation-induced Puma expression and apoptosis, we generated wild-type (WT), p53−/−, and FoxO3a−/− FDM cells and found that p53−/− but not FoxO3a−/− cells were protected against IL-3 withdrawal. Loss of p21cip/waf, which is critical for p53-mediated cell-cycle arrest, afforded no protection against IL-3 deprivation. A survival advantage was also observed in untransformed p53−/− hematopoietic progenitor cells cultured in the presence or absence of cytokines. In response to IL-3 deprivation, increased Puma protein levels in p53−/− cells were substantially delayed compared with WT cells. Increased p53 transcriptional activity was detected after cytokine deprivation. This was substantially less than that induced by DNA damage and associated not with increased p53 protein levels but with loss of the p53 regulator, MDM2. Thus, we conclude that p53 protein is activated after IL-3 deprivation by loss of MDM2. Activated p53 transcriptionally up-regulates Puma, which initiates apoptosis.

Single-cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response.

Understanding how the immune system decides between tolerance and activation by antigens requires addressing cytokine regulation as a highly dynamic process. We quantified the dynamics of interleukin-2 (IL-2) signaling in a population of T cells during an immune response by combining in silico modeling and single-cell measurements in vitro. We demonstrate that IL-2 receptor expression levels vary widely among T cells creating a large variability in the ability of the individual cells to consume, produce and participate in IL-2 signaling within the population. Our model reveals that at the population level, these heterogeneous cells are engaged in a tug-of-war for IL-2 between regulatory (T(reg)) and effector (T(eff)) T cells, whereby access to IL-2 can either increase the survival of T(eff) cells or the suppressive capacity of T(reg) cells. This tug-of-war is the mechanism enforcing, at the systems level, a core function of T(reg) cells, namely the specific suppression of survival signals for weakly activated T(eff) cells but not for strongly activated cells. Our integrated model yields quantitative, experimentally validated predictions for the manipulation of T(reg) suppression.

MAC inhibitors suppress mitochondrial apoptosis

MAC (mitochondrial apoptosis-induced channel) forms in the mitochondrial outer membrane and unleashes cytochrome c to orchestrate the execution of the cell. MAC opening is the commitment step of intrinsic apoptosis. Hence closure of MAC may prevent apoptosis. Compounds that blocked the release of fluorescein from liposomes by recombinant Bax were tested for their ability to directly close MAC and suppress apoptosis in FL5.12 cells. Low doses of these compounds (IC50 values ranged from 19 to 966 nM) irreversibly closed MAC. These compounds also blocked cytochrome c release and halted the onset of apoptotic markers normally induced by IL-3 (interleukin-3) deprivation or staurosporine. Our results reveal the tight link among MAC activity, cytochrome c release and apoptotic death, and indicate this mitochondrial channel is a promising therapeutic target.

Puma indirectly activates Bax to cause apoptosis in the absence of Bid or Bim

Bcl-2 family members regulate apoptosis in response to cytokine withdrawal and a broad range of cytotoxic stimuli. Pro-apoptotic Bcl-2 family members Bax and Bak are essential for apoptosis triggered by interleukin-3 (IL-3) withdrawal in myeloid cells. The BH3-only protein Puma is critical for initiation of IL-3 withdrawal-induced apoptosis, because IL-3-deprived Puma(-/-) cells show increased capacity to form colonies when IL-3 is restored. To investigate the mechanisms of Puma-induced apoptosis and the interactions between Puma and other Bcl-2 family members, we expressed Puma under an inducible promoter in cells lacking one or more Bcl-2 family members. Puma rapidly induced apoptosis in cells lacking the BH3-only proteins, Bid and Bim. Puma expression resulted in activation of Bax, but Puma killing was not dependent on Bax or Bak alone as Puma readily induced apoptosis in cells lacking either of these proteins, but could not kill cells deficient for both. Puma co-immunoprecipitated with the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1 but not with Bax or Bak. These data indicate that Puma functions, in the context of induced overexpression or IL-3 deprivation, primarily by binding and inactivating anti-apoptotic Bcl-2 family members.

Regulated Hox Gene Expression Reveals a Novel Role for Hox Genes in Myeloid Cell Proliferation and Survival

The Hox family of homeodomain transcription factors are essential for the regulation of hematopoiesis and deregulated expression of some Hox gene is associated with the development of myeloproliferative disorders and leukaemia. In mammals, 39 Hox genes are organized into four clusters (A, B, C or D). The expression of these genes is tightly regulated at particular differentiation points in haematopoiesis. Importantly, the over-expression of Hox genes, HoxB4, HoxA9 and HoxA10 is frequent in acute myeloid leukaemia, and may arise as a result of MLL rearrangements or from translocations fusing Hox genes to the nucleoporin Nup98. Overexpression of murine HoxB8 together with IL-3 results in myeloid leukaemia in mice. Primary myeloid progenitor cells can be immortalised using retroviral expression of Homeobox genes HoxB8 or HoxA9 in the presence of exogenous growth factors Interleukin-3 (IL-3) or GM-CSF. We have exploited this observation to generate IL-3 dependent cell lines from gene-deleted mice to identify which members of the Bcl-2 family of apoptosis regulators are required for apoptosis provoked by IL-3 deprivation (Blood, 2006 108:1461-8). Using a unique lentiviral expression system we have now generated IL-3 dependent myeloid progenitor cell lines in which we can regulate the expression of wild-type (untagged) HoxB8 or HoxA9 using 4-hydroxy tamoxifen (4HT), to determine how these genes immortalise myeloid cells. The mechanisms of action of Hox proteins in leukaemiagenesis remain to be determined but are thought, in part at least, to result from a block in myeloid differentiation. Conditional (growth-factor dependent) immortalisation of myeloid progenitors was possible only in the presence of induced Hox gene expression and surprisingly, withdrawal of HoxB8 expression did not result in terminal differentiation of all cells. Instead, loss of Hox expression, even in the presence of IL-3, induced Go/G1 cell cycle arrest and caspase-dependent cell death. This death was substantially slower that that induced by IL-3 deprivation, indicating that for some time at least, survival signals transduced by IL-3 remained intact. Thus whilst the IL-3 survival signal persisted, the proliferative signal was inhibited. We also show that HoxB8 regulates expression of the pro-apoptotic Bcl-2 family member Bim, since loss of HoxB8 resulted in substantially increased Bim expression and the cell death induced by loss of HoxB8 expression was inhibited in Bim-deficient cells. Importantly, re-addition of 4HT to cell cultures after various periods of no HoxB8 expression restored HoxB8 expression and resulted in an increase in cell viability, cell proliferation and decrease of Bim expression, indicating that at least some cells without HoxB8 expression have not terminally differentiated and retain the ability to proliferate. Our results suggest that overexpression of Hox genes such as HoxB8 (or HoxA9) contribute to myeloid transformation by coupling a growth factor signal to proliferation and also regulate the apoptotic machinery. Using this system will be able to provide proof of principal that leukemia-associated Hox genes are valid therapeutic targets.

Role of Lipocalin 24p3 in Apoptosis and Leukemia.

Programmed cell death or Apoptosis, is a critical aspect of normal physiology as well as the genesis and treatment of cancer. Certain apoptotic pathways are transcriptionally regulated; in these cases, apoptosis is induced by the transcriptional activation of genes encoding proapoptotic proteins. We originally identified lipocalin 24p3 as the gene undergoing maximum transcriptional stimulation following induction of apoptosis by cytokine-deprivation of interleukin 3 (IL-3) dependent cells. 24p3 is a member of the lipocalin family of carrier proteins – a group of small-secreted molecules that bind and transport low-molecular weight ligands. By delivering this cargo via cell-surface receptors they are known to influence many responses. 24p3 is a secreted lipocalin, which we have found induces apoptosis when added to a variety of lymphoid cells. These and other results revealed a model in which IL-3 deprivation activates 24p3 transcription, leading to synthesis and secretion of 24p3, which induces apoptosis through an autocrine/paracrine pathway. We have isolated the 24p3 cell surface receptor (24p3R) and found that 24p3 induces apoptosis through a novel pathway culminating in a decrease in intracellular iron levels (a biological iron chelator). Interestingly, iron chelators inhibit cellular proliferation and induce apoptosis, and are under active investigation as chemotherapeutic agents. The basis by which decreased intracellular iron induces apoptosis is not well understood. We performed expression-profiling experiments to identify differentially regulated genes in 24p3 and as a control in Deferoxamine (DFO), a synthetic iron chelator, treated cells. Our preliminary results suggest that 24p3 activates the expression of a novel gene, ING-2 (inhibitor of growth-2). ING-2 prevents cell growth by inducing cell cycle arrest at the G2/M phase. In contrast, the synthetic iron chelator, DFO activates the expression of NDRG1 (n-Myc downstream-regulated gene 1), which induces cell cycle arrest at G0/G1 phase. These results suggest that 24p3 induces cell death by activating regulators of the cell cycle. Finally, we have also found that the oncogene BCR-ABL counteracts the 24p3 proapoptotic pathway by misregulating expression of 24p3 and 24p3R. To study the contribution of 24p3 apoptotic pathway in the progression of CML, we have performed CML modeling experiments in mice. BCR-ABL transformed 24p3 deficient bone marrow cells failed to induce myeloproliferative disease in recipients upon transplantation. However, wild-type bone marrow cells when transduced with BCR-ABL oncogene readily induced CML-like disease in transplanted mice. Therefore, the secretion of 24p3 by BCR-ABL transformed cells facilitates the progression of CML. We have also demonstrated that 24p3 plays an important role in Gleevec-induced cell death in BCR-ABL transformed cells. These studies have therapeutic implications for Gleevec resistant CML.

Survival Signaling by C-RAF: Mitochondrial Reactive Oxygen Species and Ca2+ Are Critical Targets

Survival signaling by RAF occurs through largely unknown mechanisms. Here we provide evidence for the first time that RAF controls cell survival by maintaining permissive levels of mitochondrial reactive oxygen species (ROS) and Ca(2+). Interleukin-3 (IL-3) withdrawal from 32D cells resulted in ROS production, which was suppressed by activated C-RAF. Oncogenic C-RAF decreased the percentage of apoptotic cells following treatment with staurosporine or the oxidative stress-inducing agent tert-butyl hydroperoxide. However, it was also the case that in parental 32D cells growing in the presence of IL-3, inhibition of RAF signaling resulted in elevated mitochondrial ROS and Ca(2+) levels. Cell death is preceded by a ROS-dependent increase in mitochondrial Ca(2+), which was absent from cells expressing transforming C-RAF. Prevention of mitochondrial Ca(2+) overload after IL-3 deprivation increased cell viability. MEK was essential for the mitochondrial effects of RAF. In summary, our data show that survival control by C-RAF involves controlling ROS production, which otherwise perturbs mitochondrial Ca(2+) homeostasis.

Regulation of Phosphorylation of Thr-308 of Akt, Cell Proliferation, and Survival by the B55α Regulatory Subunit Targeting of the Protein Phosphatase 2A Holoenzyme to Akt

Akt is a protein serine/threonine kinase that is involved in the regulation of diverse cellular processes. Phosphorylation of Akt at regulatory residues Thr-308 and Ser-473 leads to its full activation. The protein phosphatase 2A (PP2A) has long been known to negatively regulate Akt activity. The PP2A holoenzyme consists of the structural subunit (A), catalytic subunit (C), and a variable regulatory subunit (B). Here we report the identification of the specific B regulatory subunit that targets the PP2A holoenzyme to Akt. We found endogenous association of PP2A AB55C holoenzymes with Akt by co-immunoprecipitation analyses in pro-lymphoid FL5.12 cells. Akt was shown to associate with ectopically expressed B55alpha subunit in NIH3T3 cells. The direct interaction between B55alpha subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55alpha subunit significantly impaired phosphorylation at Thr-308, but to a lesser extent at Ser-473 of Akt in both FL5.12 and NIH3T3 cells. Concomitantly, phosphorylation of a subset of Akt substrates, including FoxO3a, was substantially decreased by B55alpha overexpression in these cells. Silencing of B55alpha expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55alphaC holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55alpha overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55alpha expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55alpha-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival.

HTLV-I infection of WE17/10 CD4+ cell line leads to progressive alteration of Ca2+ influx that eventually results in loss of CD7 expression and activation of an antiapoptotic pathway involving AKT and BAD which paves the way for malignant transformation

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy slowly emerging from human T-cell leukemia virus type 1 (HTLV-I)-infected mature CD4(+) T-cells. To characterize the molecular modifications induced by HTLV-I infection, we compared HTLV-I-infected WE17/10 cells with control cells, using micro-arrays. Many calcium-related genes were progressively downmodulated over a period of 2 years. Infected cells acquired a profound decrease of intracellular calcium levels in response to ionomycin, timely correlated with decreased CD7 expression. Focusing on apoptosis-related genes and their relationship with CD7, we observed an underexpression of most antiapoptotic genes. Western blotting revealed increasing Akt and Bad phosphorylation, timely correlated with CD7 loss. This was shown to be phosphatidylinositol 3-kinase (PI3K)-dependent. Activation of PI3K/Akt induced resistance to the apoptotic effect of interleukin-2 deprivation. We thus propose the following model: HTLV-I infection induces a progressive decrease in CD3 genes expression, which eventually abrogates CD3 expression; loss of CD3 is known to perturb calcium transport. This perturbation correlates with loss of CD7 expression and induction of Akt and Bad phosphorylation via activation of PI3K. The activation of the Akt/Bad pathway generates a progressive resistance to apoptosis, at a time HTLV-I genes expression is silenced, thus avoiding immune surveillance. This could be a major event in the process of the malignant transformation into ATLL.

A Cell-Surface Receptor for Lipocalin 24p3 Selectively Mediates Apoptosis and Iron Uptake

The lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis due to interleukin-3 (IL-3) deprivation and iron transport. Here we report cloning of the 24p3 cell-surface receptor (24p3R). Ectopic 24p3R expression confers on cells the ability to undergo either iron uptake or apoptosis, dependent upon the iron content of the ligand: Iron-loaded 24p3 increases intracellular iron concentration without promoting apoptosis; iron-lacking 24p3 decreases intracellular iron levels, which induces expression of the proapoptotic protein Bim, resulting in apoptosis. Intracellular iron delivery blocks Bim induction and suppresses apoptosis due to 24p3 addition or IL-3 deprivation. We find, unexpectedly, that the BCR-ABL oncoprotein activates expression of 24p3 and represses 24p3R expression, rendering BCR-ABL(+) cells refractory to secreted 24p3. By inhibiting BCR-ABL, imatinib induces 24p3R expression and, consequently, apoptosis. Our results reveal an unanticipated role for intracellular iron regulation in an apoptotic pathway relevant to BCR-ABL-induced myeloproliferative disease and its treatment.