Protein kinase C in cellular transformation: a valid target for therapy?

The protein kinase C (PKC) family of serine/threonine protein kinases share structural homology, while exhibiting substantial functional diversity. PKC isoforms are ubiquitously expressed in tissues which makes it difficult to define roles for individual isoforms, with complexity compounded by the finding that PKC isoforms can co-operate with or antagonize other PKC family members. A number of studies suggest the involvement of PKC family members in regulating leukaemic cell survival and proliferation. Chronic lymphocytic leukaemia (CLL), the most common leukaemia in the Western world, exhibits dysregulated expression of PKC isoforms, with recent reports indicating that PKCβ and δ play a critical role in B-cell development, due to their ability to link the B-cell receptor (BCR) with downstream signalling pathways. Given the prognostic significance of the BCR in CLL, inhibition of these BCR/PKC-mediated signalling pathways is of therapeutic relevance. The present review discusses the emerging role of PKC isoforms in the pathophysiology of CLL and assesses approaches that have been undertaken to modulate PKC activity

The role of mTOR-mediated signaling in the regulation of cellular migration

Mechanistic target for rapamycin (mTOR) is a serine/threonine protein kinase that forms two distinct complexes mTORC1 and mTORC2, integrating mitogen and nutrient signals to regulate cell survival and proliferation; processes which are commonly deregulated in human cancers. mTORC1 and mTORC2 have divergent molecular associations and cellular functions: mTORC1 regulates in mRNA translation and protein synthesis, while mTORC2 is involved in the regulation of cellular survival and metabolism. Through AKT phosphorylation/activation, mTORC2 has also been reported to regulate cell migration. Recent attention has focused on the aberrant activation of the PI3K/mTOR pathway in B cell malignancies and there is growing evidence for its involvement in disease pathogenesis, due to its location downstream of other established novel drug targets that intercept B cell receptor (BCR) signals. Shared pharmacological features of BCR signal inhibitors include a striking “lymphocyte redistribution” effect whereby patients experience a sharp increase in lymphocyte count on initiation of therapy followed by a steady decline. Chronic lymphocytic leukemia (CLL) serves as a paradigm for migration studies as lymphocytes are among the most widely travelled cells in the body, a product of their role in immunological surveillance. The subversion of normal lymphocyte movement in CLL is being elucidated; this review aims to describe the migration impairment which occurs as part of the wider context of cancer cell migration defects, with a focus on the role of mTOR in mediating migration effects downstream of BCR ligation and other microenvironmental signals

Nfix expression critically modulates early B lymphopoiesis and myelopoiesis

The commitment of stem and progenitor cells toward specific hematopoietic lineages is tightly controlled by a number of transcription factors that regulate differentiation programs via the expression of lineage restricting genes. Nuclear factor one (NFI) transcription factors are important in regulating hematopoiesis and here we report an important physiological role of NFIX in B- and myeloid lineage commitment and differentiation. We demonstrate that NFIX acts as a regulator of lineage specification in the haematopoietic system and the expression of Nfix was transcriptionally downregulated as B cells commit and differentiate, whilst maintained in myeloid progenitor cells. Ectopic Nfix expression in vivo blocked early B cell development stage, coincident with the stage of its downregulation. Furthermore, loss of Nfix resulted in the perturbation of myeloid and lymphoid cell differentiation, and a skewing of gene expression involved in lineage fate determination. Nfix was able to promote myeloid differentiation of total bone marrow cells under B cell specific culture conditions but not when expressed in the hematopoietic stem cell (HSPC), consistent with its role in HSPC survival. The lineage choice determined by Nfix correlated with transcriptional changes in a number of genes, such as E2A, C/EBP, and Id genes. These data highlight a novel and critical role for NFIX transcription factor in hematopoiesis and in lineage specification

In Vivo Detection of Intracellular Signaling Pathways in Developing Thymocytes

Information regarding the intracellular signaling processes that occur during the development of T cells has largely been obtained with the use of transgenic mouse models, which although providing invaluable information are time consuming and costly. To this end, we have developed a novel system that facilitates the In Vivo analysis of signal transduction pathways during T-lymphocyte development. This approach uses reporter-plasmids for the detection of intracellular signals mediated by the mitogen-activated protein kinase or cyclic AMP-dependent protein kinase. Reporter-plasmids are transfected into thymocytes in fetal thymic organ culture by accelerated DNA/particle bombardment (gene gun), and the activation of a signaling pathway is determined in the form of a standard luciferase assay. Importantly, this powerful technique preserves the structural integrity of the thymus, and will provide an invaluable tool to study how thymocytes respond to normal environmental stimuli encountered during differentiation within the thymic milieu. Thus, this method allows for the monitoring of signals that occur in a biological time frame, such as during differentiation, and within the natural environment of differentiating cells

The differential regulation of cyclic AMP phosphodiesterases in T lymphocytes

Cyclic AMP phosphodiesterases were studied in T lymphocytes from different maturation stages, tissues and species. Although cyclic AMP phosphodiesterases were characterised in lymphocytes isolated from murine spleens, human tonsils, a murine thymoma cell line and a human mature T cell line, the main focus of this study was cyclic AMP PDE activities from murine thymocytes. With the use of selective phosphodiesterase inhibitors, the relative contribution that particular phosphodiesterase isoforms made towards total phosphodiesterase activity in the cells was assessed. Thus, cyclic GMP stimulated, PDE2 activity and cyclic AMP specific PDE4 activity were shown to provide the major cyclic AMP hydrolysing activities in murine thymocytes. PDE4 activity predominated (~80% total) in the absence of cyclic GMP. However, on addition of low (10?M) concentrations of cyclic GMP, PDE2 activity constituted the major PDE activity in thymocytes (~80% total). The PDE4 selective inhibitor rolipram inhibited murine thymocyte PDE4 activity in a dose-dependent manner (IC50 ~65nM). PDE2 activity was stimulated in a dose-dependent fashion (EC50 ~l?M) by cyclic GMP and was selectively inhibited by the compound, EHNA (IC50 ~4?M). No calcium/calmodulin stimulated, PDEl activity or cyclic GMP-inhibited, PDE3 activity was found to be present in murine thymocytes. The non-selective PDE inhibitor IBMX elicited >98% inhibition of PDE activity in thymocytes indicating the absence of IBMX- insensitive, PDE7 activity in these cells. FPLC analysis confirmed these findings, revealing a rolipram inhibited PDE4 activity and a cyclic GMP stimulated, EHNA inhibited, PDE2 activity, but no PDEl, PDE3 or PDE7 activity. The selective inhibitors identified the profile of phosphodiesterase activities in T cells from other sources. Thus, PDE2 activity was also found to be present in murine splenic lymphocytes, proliferating T3.2 thymoma cell line derived from murine thymus and the human leukemic T cell line, Jurkat. In contrast to murine thymocytes, PDE3 appeared to be present in human tonsillar T lymphocytes, the human T cell line Jurkat and the thymoma cell line, T3.2. Similar to murine thymocytes, PDE4 activities were found to be present in the murine thymoma cell line, T3.2 and the human T cell line, Jurkat. Moreover, primary lymphocytes isolated from murine thymuses, spleens and human tonsils did not contain Ca2+/CaM stimulated, PDE1 activity. In contrast, the proliferating human Jurkat T cell line contained calcium/calmodulin-stimulated PDEl and an IBMX insensitive PDE activity. The differential distributions of phosphodiesterase activities within these lymphoid cells indicated species and possibly maturation-dependent differences of phosphodiesterase expression within these cells. Upon ligation of the TCR/CD3 complex with either the mitogenic lectin phytohaemagluttinin or anti-TCR/anti-CD3 monoclonal antibodies, phosphodiesterase activities and cyclic AMP levels were found to be rapidly and differentially regulated in murine thymocytes. Within 5 minutes of challenge with PHA, there was a transient decrease (~83%) in PDE4 activity and in PDE2 activity (~40%), returning almost to basal in 20 minutes. Both anti-CD3 and anti-TCR antibodies also caused an initial reduction in the PDE4 activity (~50%) which was then followed by a sustained increase in activity. In contrast to that observed with PHA, anti-TCR/CD3 antisera had little effect on PDE2 activity. Cyclic AMP levels were also differentially regulated upon stimulation of thymocytes with either PHA or anti-CD3 monoclonal antibodies. Hence, there was a transient 2-fold elevation in cyclic AMP levels upon stimulation of thymocytes with PHA, which reached basal levels again within 30 minutes. In contrast anti-CD3 antibody stimulation of thymocytes led to a slow sustained elevation in cyclic AMP levels over a 30 minute period. Study of the mechanism controlling the anti-CD3-stimulated elevation in PDE4 activity in murine thymocytes revealed that the increase in this phosphodiesterase activity was dependent on protein tyrosine kinase and protein kinase C-mediated signals. This interpretation was based on findings that the tyrosine kinase inhibitor, genistein partially inhibited the TCR-mediated elevation in PDE4 activity. Moreover, the protein kinase C selective inhibitor chelerythrine blocked the rise of PDE4 activity and the phorbol ester, phorbol-12-myristate, 13- acetate caused an increase in PDE4 activity which was of a similar magnitude to the elevation seen in the presence of anti-CD3 antibodies. Interestingly, the elevation in PDE4 activity was blocked upon stimulation of thymocytes with TPA or anti-CD3 antibody in the presence cyclohexamide or actinomycin D. This indicated that a rapid induction of PDE4 activity occurred in murine thymocytes upon crosslinking of the TCR/CD3 complex. These data identify 'crosstalk' between cyclic AMP and PKC mediated signalling pathways

The role of mTOR-mediated signals during haemopoiesis and lineage commitment

The serine/threonine protein kinase mechanistic target of rapamycin (mTOR) has been implicated in the regulation of an array of cellular functions including protein and lipid synthesis, proliferation, cell size and survival. Here, we describe the role of mTOR during haemopoiesis within the context of mTORC1 and mTORC2, the distinct complexes in which it functions. The use of conditional transgenic mouse models specifically targeting individual mTOR signalling components, together with selective inhibitors, have generated a significant body of research emphasising the critical roles played by mTOR, and individual mTOR complexes, in haemopoietic lineage commitment and development. This review will describe the profound role of mTOR in embryogenesis and haemopoiesis, underscoring the importance of mTORC1 at the early stages of haemopoietic cell development, through modulation of stem cell potentiation and self-renewal, and erythroid and B cell lineage commitment. Furthermore, the relatively discrete role of mTORC2 in haemopoiesis will be explored during T cell development and B cell maturation. Collectively, this review aims to highlight the functional diversity of mTOR signalling and underline the importance of this pathway in haemopoiesis

Evidence that hematopoietic stem cell function is preserved during aging in long-lived S6K1 mutant mice

The mechanistic target of rapamycin (mTOR) signalling pathway plays a highly conserved role in aging; mice lacking ribosomal protein S6 kinase 1 (S6K1-/-) have extended lifespan and healthspan relative to wild type (WT) controls. Exactly how reduced mTOR signalling induces such effects is unclear, although preservation of stem cell function may be important. We show, using gene expression analyses, that there was a reduction in expression of cell cycle genes in young (12 week) and aged (80 week) S6K1-/- BM-derived c-Kit+ cells when compared to age-matched WT mice, suggesting that these cells are more quiescent in S6K1-/- mice. In addition, we investigated hematopoietic stem cell (HSC) frequency and function in young and aged S6K1-/- and WT mice. Young, but not aged, S6K1-/- mice had more LSK (lineage-, c-Kit+, Sca-1+) cells (% of bone marrow (BM)), including the most primitive long-term repopulating HSC (LT-HSC) relative to WT controls. Donor-derived engraftment of LT-HSCs in recipient mice was unaffected by genotype in young mice, but was enhanced in transplants using LT-HSCs derived from aged S6K1-/- mice. Our results are the first to provide evidence that age-associated HSC functional decline is ameliorated in a long-lived mTOR mutant mouse

Targeting proliferating CLL cells with a novel synthetic low density lipoprotein drug delivery system

Chronic lymphocytic leukaemia (CLL) currently remains incurable without stem cell transplantation, an option for only the minority of patients. Despite advances in chemotherapy, most patients relapse owing to the persistence of minimal residual disease (MRD). Substantial evidence has accrued to suggest that the tumour microenvironment is central to disease progression in CLL, with the bone marrow (BM) and lymph nodes (LN) acting as sanctuary sites for MRD. Whilst peripheral blood CLL cells are cell cycle arrested, significant rates of clonal proliferation occur in the BM/LN wherein acquisition of deleterious cytogenetic abnormalities such as 17p deletion may arise. Further, CLL cells co-cultured in vitro on stroma with CD154/IL-4 to give a proliferative signal, are chemoresistant to first line therapies. As proliferating cells require lipids for membrane synthesis, we hypothesise that proliferating CLL cells will have greater requirement for low density lipoprotein (LDL) compared to circulating CLL cells, and also that of normal resting lymphocytes providing a potentially differential cellular property to attack. Proof of concept of drug-loaded synthetic (s)LDL nanoparticles has been provided in glioblastoma and CML. We propose that drug loading into sLDL nanoparticles will allow selective targeting of proliferating CLL cells within the BM/LN proliferation centre, will protect drugs from plasma binding proteins, and will ultimately raise intracellular drug concentrations in the protective microenvironmental niche, to overcome chemoresistance. Aims. To determine (a) the extent of sLDL uptake by CLL cells compared to normal; and (b) whether sLDL uptake by CLL cells changes under proliferative conditions mimicking the proliferation centre. This will determine whether proliferating CLL cells have increased sLDL uptake compared to non-cycling CLL cells or normal B lymphocytes. Methods. sLDL uptake was assessed by flow cytometry, measuring the mean fluorescence intensity in the FITC channel owing to the stable incorporation of dioctadecyloxacarbocyanine (DiO) into the formulation. Internalisation was confirmed by deconvolution fluorescence microscopy. Primary CLL and normal donor samples were enriched for CD19+ B-lineage cells by magnetically activated cell sorting. Cells were cultured in media on tissue culture plastic or NT-L mouse fibroblasts with or without CD154/IL4. Lymphoid cells were stained with CellTrace VioletR to track cell division in response to proliferative signals (CD154/IL4 stroma). Results. HG3, a human lymphoblastoid cell line, avidly took up sLDL nanoparticles in a concentration (0-50 ng/mL cholesterol) and time (0.5-24h) dependent manner. Normal donor peripheral blood B-cells and CLL cells cultured on plastic did not actively take up sLDL but maintained their viability even in the highest concen- tration sLDL tested. Actively proliferating CLL cells on CD154/IL4 stroma could be targeted with sLDL unlike their non-cycling counterparts; interestingly even the minor population of cells that had remained undivided on stroma were also found to be sLDL positive. Summary. CLL cells can be selectively targeted by sLDL nanoparticles with respect to their non-cycling counterparts. We next will investigate the in vivo targeting of sLDL which we hypothesise, by virtue of their size, will home to lymphoreticular organs, sanctuary sites for CLL MRD

Generation of a poor prognostic chronic lymphocytic leukemia-like disease model: PKC subversion induces up-regulation of PKC II expression in B lymphocytes

Overwhelming evidence identifies the microenvironment as a critical factor in the development and progression of chronic lymphocytic leukemia, underlining the importance of developing suitable translational models to study the pathogenesis of the disease. We previously established that stable expression of kinase dead protein kinase C alpha in hematopoietic progenitor cells resulted in the development of a chronic lymphocytic leukemia-like disease in mice. Here we demonstrate that this chronic lymphocytic leukemia model resembles the more aggressive subset of chronic lymphocytic leukemia, expressing predominantly unmutated immunoglobulin heavy chain genes, with upregulated tyrosine kinase ZAP-70 expression and elevated ERK-MAPK-mTor signaling, resulting in enhanced proliferation and increased tumor load in lymphoid organs. Reduced function of PKCα leads to an up-regulation of PKCβII expression, which is also associated with a poor prognostic subset of human chronic lymphocytic leukemia samples. Treatment of chronic lymphocytic leukemia-like cells with the selective PKCβ inhibitor enzastaurin caused cell cycle arrest and apoptosis both in vitro and in vivo, and a reduction in the leukemic burden in vivo. These results demonstrate the importance of PKCβII in chronic lymphocytic leukemia-like disease progression and suggest a role for PKCα subversion in creating permissive conditions for leukemogenesis

Subcellular fractionation of primary chronic lymphocytic leukemia cells to monitor nuclear/ cytoplasmic protein trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data