Induction of apoptosis and CD10/neutral endopeptidase expression by jaspamide in HL-60 line cells

Jaspamide (jasplakinolide) is a natural peptide isolated from marine sponges of Jaspis species and has fungicidal and growth-inhibiting activities. We characterized the jasplakinolide-induced loss of viability by programmed cell death in the HL-60 human promyelocytic leukemia cell line and found that this process was accompanied by neutral endopeptidase (NEP)/CD10 expression on the surface of the apoptotic cells. HL-60 cells do not normally express detectable amounts of NEP/CD10 on their surface or intracytoplasmically, but upon jaspamide treatment, CD10 was synthesized de novo, its expression being inhibited by cycloheximide pretreatment. Once synthesized, NEP/CD10 interfered with the jasplakinolide signal delivered to HL-60 cells. Inhibition of NEP/CD10 by the NEP inhibitor phosphoramidon or by an anti-CD10 monoclonal antibody significantly increased apoptosis induction. The appearance of CD10 on the cell surface was blocked by preincubation of the cells with the monocytic/macrophage-differentiating agents vitamin D3 and phorbol 12-myristate 13-acetate, but not by the granulocytic differentiating agents retinoic acid or dimethyl sulfoxide. Moreover, in the promonocytic U937 and mature monocytic THP-1 cell lines, jaspamide induced apoptosis but not CD10 expression. In HL-60 cells, CD10 expression was partially but not totally blocked by the broad-spectrum caspase inhibitor benzyloxacarbonyl-Val-Ala-Asp-fluoromethylketone, indicating a connection between apoptosis induction and CD10 synthesis. Our findings suggest that the CD10 expression is related to the programmed cell death induction by jaspamide, and also with the process of granulocytic differentiation in HL-60 cells. Received 22 April 2002; received after revision 8 June 2002; accepted 10 June 2002     

ERKs are the point of divergence of PKA and PKC activation by PTHrP in human skin fibroblasts

Parathyroid hormone-related peptide (PTHrP) receptors, coupled to trimeric G proteins, operate in most target cells through at least three different transduction routes: Gαs-mediated stimulation of adenylylcyclase (AC), Gαq-mediated activation of phospholipase Cβ (PLC) and mitogen-activated protein kinase (MAPK) activation. In this study we investigated the relative role of different pathways in human skin fibroblast prolifera-tion. Using chemical inhibitors and activators of signal transduction, we demonstrated that: (i) AC/cAMP and PLC/1,4,5 inositol triphosphate/diacylglycerol second-messenger systems are simultaneously activated following PTHrP binding to its receptors; (ii) the mitogenic response to PTHrP derives from a balance between two counteracting pathways – an activating route mediated by protein kinase C (PKC) and an inhibitory route mediated by protein kinase A (PKA); (iii) PTHrP mitogenic effects are largely dependent on MAPKs, whose activity can be modulate d by both PKA and PKC. Our results indicate that MAPKs are common targets of both transduction routes and, at the same time, their point of divergence in mediating PTHrP dual and opposite mitogenic effects. Received 2 August 2002; received after revision 10 September 2002; accepted 18 October 2002 RID="*" ID="*"Corresponding author.     

An anti-Bcl-2 antibody prevents 2-deoxy-D-ribose-induced apoptosis in the IPLB-LdFB insect cell line

Confocal microscopy reveals that the anti-Bcl-2 antibody (pAb) is able to diffuse across the plasma membrane of the fat body cell line IPLB-LdFB from the insect Lymantria dispar, demonstrating the presence of Bcl-2-like molecules in the cytoplasm. Immunoperoxidase procedure confirms the cellular localization. Furthermore, an immunoprecipitation corresponding to a molecular weight of 29 KDa is observed with western blot analysis using the anti-Bcl-2 pAb. Cytofluorimetric experiments show that anti-Bcl-2 pAb counteracts 2-deoxy-d-ribose-induced apoptosis and provokes morphological changes in the insect cell line, i.e. a reduction in cell size, the disappearance of the vacuola and changes in shape. At the same time, the antibody provokes mitochondrial membrane depolarization, and N-acetyl-l-cysteine is unable to reconstitute the physiological conditions. The present findings suggest that Bcl-2-like proteins play a main role in maintaining of the integrity of cellular components, e.g. mitochondria, rather than in controlling programmed cell death. Received 23 January 2001; received after revision 1 March 2001; accepted 1 March 2001     

Protein kinases: which one is the memory molecule?

Encoding of new experiences is likely to induce activity-dependent modifications in the brain. Studies in organisms far apart on the phylogenetic scale have shown that similar, sometimes identical, signal transduction pathways subserve plasticity in neuronal systems, and they may play pivotal roles in the formation of long-term memories. It has become evident that phosphorylation/dephosphorylation reactions are critical for the initiation of cellular mechanisms that embody, retain and modify information in neural circuits. Although physiological investigations on synaptic plasticity have had a major impact, we have concentrated our review on behavioural studies that provide direct or indirect evidence for a role of kinases in mechanisms underlying memory formation. From these, it appears that the learning event induces activation of a variety of kinases with specific time courses. For instance, the calcium/calmodulin-dependent protein kinase II seems to participate in an early phase of memory formation. Apparently, activation of both protein tyrosine kinases and mitogen-activated protein kinases is required for much longer and may thus have a particular function during transformation from short-term into long-term memory. Quite different time courses appear for protein kinase C (PKC) and protein kinase A (PKA), which may function at two different time points, shortly after training and again much later. This suggests that PKC and PKA might play a role at early and late stages of memory formation. However, we have considered some examples showing that these signalling pathways do not function in isolation but rather interact in an intricate intracellular network. This is indicative of a more complex contribution of each kinase to the fine tuning of encoding and information processing. To decipher this complexity, pharmacological, biochemical and genetic investigations are more than ever necessary to unravel the role of each kinase in the syntax of learning and memory formation.     

The role of thrombospondin-1 in apoptosis

The thrombospondins are a family of extracellular proteins that participate in cell-to-cell and cell-to-matrix communication. They regulate cellular phenotype during tissue genesis and repair. Five family members, each representing a separate gene product, probably exist in most vertebrate species. Like most extracellular proteins, the thrombospondins are composed of several structural domains that are responsible for the numerous biological functions that have been described for this protein family. Considerable progress has been made towards understanding the function of thrombospondins. The role of thrombospondin in the process of apoptosis or programmed cell death has recently come into focus. In this review we will concentrate on the role of thrombospondin-1 in the broad field of apoptotis research. Received 5 December 2001; received after revision 28 March 2002; accepted 28 March 2002     

The isoform-specific regulation of apoptosis by protein kinase C

The process of apoptosis is regulated at several levels through phosphorylation by many different protein kinases. The protein kinase C (PKC) family, which comprises at least 10 isoforms with distinct means of regulation and tissue distribution patterns, have been shown to exert both inhibitory and stimulatory influences on apoptosis. This review details recent progress made in determining the roles played by individual PKC isoforms in the control of apoptosis, with reference to their target substrates and actions in different cell types. Although notable exceptions exist, the weight of evidence indicates that the alpha, beta, epsilon and atypical isoforms are anti-apoptotic in their action, whereas the delta and theta isoforms are usually involved in the promotion of apoptosis.     

Apolipoprotein CIII hyperactivates β cell CaV1 channels through SR-BI/β1 integrin-dependent coactivation of PKA and Src

Apolipoprotein CIII (ApoCIII) not only serves as an inhibitor of triglyceride hydrolysis but also participates in diabetes-related pathological events such as hyperactivation of voltage-gated Ca²⁺ (Ca_V) channels in the pancreatic β cell. However, nothing is known about the molecular mechanisms whereby ApoCIII hyperactivates β cell Ca_V channels. We now demonstrate that ApoCIII increased Ca_V1 channel open probability and density. ApoCIII enhanced whole-cell Ca²⁺ currents and the Ca_V1 channel blocker nimodipine completely abrogated this enhancement. The effect of ApoCIII was not influenced by individual inhibition of PKA, PKC, or Src. However, combined inhibition of PKA, PKC, and Src counteracted the effect of ApoCIII, similar results obtained by coinhibition of PKA and Src. Moreover, knockdown of β1 integrin or scavenger receptor class B type I (SR-BI) prevented ApoCIII from hyperactivating β cell Ca_V channels. These data reveal that ApoCIII hyperactivates β cell Ca_V1 channels through SR-BI/β1 integrin-dependent coactivation of PKA and Src.     

Survival of docetaxel-resistant prostate cancer cells in vitro depends on phenotype alterations and continuity of drug exposure

We evaluated in vitro the effect of paclitaxel and docetaxel on PC-3 and DU-145 prostate cancer cell lines to understand better the downstream events in drug-induced tumor cell death. Taxane treatments of DU-145 cells induced rapid cell death by apoptosis, but in PC-3 cells, treatments achieved growth arrest, followed by extensive karyokinesis resulting in multinucleation, giant-cell formation and delayed cell death. To determine if the giant multinucleated cells were able to produce proliferating and drug-resistant survivors, we first delineated the kinetics of drug activity and cytotoxic dose range. Analysis of both lines by colorimetric and cell viability assays demonstrated improved cytotoxicity of taxanes applied continuously. Selected doses and schedules of docetaxel were used to induce giant multinucleated cells that gave rise to docetaxel-resistant survivors, which remained sensitive to paclitaxel and other chemotherapeutics. Growth and morphology of the recovered clones was similar to parental cells. The resistant phenotype of these clones determined by immunofluorescence and immunoblot was associated with transient expression of the β-tubulin IV isoform and was independent of P-glycoprotein, bcl-2 and bcl-xL. Resistant clones will be useful to model progression of resistance to taxanes and to identify unknown and clinically important molecular mechanisms of cell death and resistance. Received 15 March 2002; received after revision 25 April 2002; accepted 27 May 2002     

The effect of isoforms of the cell polarity protein, human ASIP, on the cell cycle and Fas/FasL-mediated apoptosis in human hepatoma cells

Human ASIP (hASIP) is expressed as numerous alternative splicing isoforms and there is an atypical protein kinease C (aPKC) phosphorylation site in exon 17b of the encoded sequence. We have identified an important role for exon 17b in cancer cells. Our results showed that hASIP-sa and sb had different effects on cell growth and Fas/FasL-mediated apoptosis in BEL-7404 human hepatoma cells. Human ASIP-sa modified the S phase of the cell cycle and might stimulate cell proliferation. Growth inhibition by hASIP-a antisense oligonucleotide-confirmed the positive action of hASIP-sa. Compared with hASIP-sa, hASIP-sb accelerated Fas/FasL-induced apoptosis, examined by sub-G1 accumulation, chromatin condensation, nuclear fragmentation, PARP cleavage, caspase-8 degradation and mitochondria- regulated cell death. Treatment with aPKC inhibitor could enhance Fas/FasL-mediated apoptosis in hASIP-sa-overexpressing cells, suggesting that hASIP-sa and its interaction with aPKC might contribute to the malignant growth and the blocking of Fas/FasL-mediated apoptosis, while hASIP-sb might function as an antagonist of hASIP-sa.Received 24 March 2005; received after revision 31 May 2005; accepted 21 June 2005     

EGF-induced programmed cell death of human mammary carcinoma MDA-MB-468 cells is preceded by activation of AP-1

MDA-MB-468 is a human mammary adenocarcinoma cell line that overexpresses the epidermal growth factor (EGF) receptor and undergoes programmed cell death (apoptosis) in response to EGF treatment. Programmed cell death was shown to be greatly enhanced when cells were growth-arrested prior to EGF treatment. Apoptosis was characterized by an initial rounding up and detachment of the cells from their substrate starting about 12 h after EGF treatment, followed by chromatin condensation, nuclear fragmentation and oligonucleosomal fragmentation of the DNA at about 24 to 48 h. Cell death was dependent on de novo protein synthesis. We found a rapid induction of c-fos, c-jun and junB at the mRNA level after about 30 min of EGF treatment and a more delayed upregulation of fosB and fra-1. The junD gene was expressed in the absence of EGF, and it was moderately induced within 30 min of growth factor addition. The increase of the different fos and jun mRNAs were paralleled by an increase of activator protein-1 (AP-1) DNA binding activity. A characterization of the AP-1 complex revealed similar levels of several Fos and Jun proteins. Based on the kinetics of AP-1 accumulation and cell death, it seems likely that AP-1 contributes to the apoptotic cell death of EGF receptor-overexpressing MDA-MB-468 cells. Received 21 July 1997; received after revision 6 November 1997; accepted 6 November 1997     

Dual antiglioma action of metformin: cell cycle arrest and mitochondria-dependent apoptosis

The present study reports for the first time a dual antiglioma effect of the well-known antidiabetic drug metformin. In low-density cultures of the C6 rat glioma cell line, metformin blocked the cell cycle progression in G₀/G₁ phase without inducing significant cell death. In confluent C6 cultures, on the other hand, metformin caused massive induction of caspase-dependent apoptosis associated with c-Jun N-terminal kinase (JNK) activation, mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine), while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride, iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C, an inhibitor of AMP-activated protein kinase (AMPK), and was mimicked by the AMPK agonist AICAR. Similar effects were observed in the human glioma cell line U251, while rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin. Received 14 February 2007; received after revision 26 March 2007; accepted 3 April 2007     

Protein phosphatases and their potential implications in neuroprotective processes

Several neurological disorders such as stroke, amyotrophic lateral sclerosis and epilepsy result from excitotoxic events and are accompanied by neuronal cell death. These processes engage multiple signalling pathways and recruit numerous molecular components, in particular several families of protein kinases and protein phosphatases. While many investigations have examined the importance of protein kinases in excitotoxicity, protein phosphatases have not been well studied in this context. However, recent advances in understanding the functions of protein phosphatases have suggested that they may play a neuroprotective role. In this review, we summarize some of the recent findings that illustrate the pleiotropic and complex functions of tyrosine and serine/threonine protein phosphatases in the cascade of events leading to neuronal cell death, and highlight their potential intervention in limiting the extent of neuronal death.Received 8 January 2005; received after revision 3 March 2005; accepted 14 March 2005     

Mechanisms controlling cellular suicide: role of Bcl-2 and caspases

Apoptosis is an essential and highly conserved mode of cell death that is important for normal development, host defense and suppression of oncogenesis. Faulty regulation of apoptosis has been implicated in degenerative conditions, vascular diseases, AIDS and cancer. Among the numerous proteins and genes involved, members of the Bcl-2 family play a central role to inhibit or promote apoptosis. In this article, we present up-to-date information and recent discoveries regarding biochemical functions of Bcl-2 family proteins, positive and negative interactions between these proteins, and their modification and regulation by either proteolytic cleavage or by cytosolic kinases, such as Raf-1 and stress-activated protein kinases. We have critically reviewed the functional role of caspases and the consequences of cleaving key substrates, including lamins, poly(ADP ribose) polymerase and the Rb protein. In addition, we have presented the latest Fas-induced signalling mechanism as a model for receptor-linked caspase regulation. Finally, the structural and functional interactions of Ced-4 and its partial mam malian homologue, apoptosis protease activating factor-1 (Apaf-1), are presented in a model which includes other Apafs. This model culminates in a caspase/Apaf regulatory cascade to activate the executioners of programmed cell death following cytochrome c release from the mitochondria of mammalian cells. The importance of these pathways in the treatment of disease is highly dependent on further characterization of genes and other regulatory molecules in mammals. Received 18 February 1998; accepted February 1998     

Evidence of undiscovered cell regulatory mechanisms: phosphoproteins and protein kinases in mitochondria

The finding that mitochondria contain substrates for protein kinases lead to the discovery that protein kinases are located in the mitochondria of certain tissues and species. These include pyruvate dyhydrogenase kinase, branched-chain α-ketoacid dehydrogenase kinase, protein kinase A, protein kinase Cδ, stress-activated kinase and A-Raf as well as unidentified kinases. Recent evidence suggests that mitochondrial protein kinases may be involved in physiological processes such as apoptosis and steroidogenesis. Additionally, the novel finding of low-molecular-weight GTP-binding proteins in mitochondria suggests the possibility that these may interact with mitochondrial protein kinases to regulate the activity of mitochondrial effector proteins. The fact that there are components of cellular regulatory systems in mitochondria indicates the exciting possibility of undiscovered systems regulating mitochondrial physiology. Received 19 June 2001; received after revision 7 August 2001; accepted 8 August 2001     

Phosphorylation and activation of the atypical kinase p53-related protein kinase (PRPK) by Akt/PKB

p53-related protein kinase (PRPK), the human homologue of yeast Bud32, belonging to a small subfamily of atypical protein kinases, is inactive unless it is previously incubated with cell lysates. Here we show that such an activation of PRPK is mediated by another kinase, Akt/PKB, which phosphorylates PRPK at Ser250. We show that recombinant PRPK is phosphorylated in vitro by Akt and its phospho-form is recognized by a Ser250-phospho-specific antibody; that cell co-transfection with Akt along with wild-type PRPK, but not with its Ser250Ala mutant, results in increased PRPK phosphorylation; and that the phosphorylation of p53 at Ser15, the only known substrate of PRPK, is markedly increased by co-transfection of Akt with wild-type PRPK, but not PRPK dead mutant, and is abrogated by cell treatment with the Akt pathway inhibitor LY294002. Our data disclose an unanticipated mechanism by which PRPK can be activated and provide a functional link between this enigmatic kinase and the Akt signaling pathway.     

Expression of the Stp1 LMW-PTP and inhibition of protein CK2 display a cooperative effect on immunophilin Fpr3 tyrosine phosphorylation and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> growth

Although the yeast genome does not encode bona fide protein tyrosine kinases, tyrosine-phosphorylated proteins are numerous, suggesting that besides dual-specificity kinases, some Ser/Thr kinases are also committed to tyrosine phosphorylation in Saccharomyces cerevisiae. Here we show that blockage of the highly pleiotropic Ser/Thr kinase CK2 with a specific inhibitor synergizes with the overexpression of Stp1 low-molecular-weight protein tyrosine phosphatase (PTP) in inducing a severe growth-defective phenotype, consistent with a prominent role for CK2 in tyrosine phosphorylation in yeast. We also present in vivo evidence that immunophilin Fpr3, the only tyrosine-phosphorylated CK2 substrate recognized so far, interacts with and is dephosphorylated by Spt1. These data disclose a functional correlation between CK2 and LMW-PTPs, and suggest that reversible phosphorylation of Fpr3 plays a role in the regulation of growth rate and budding in S. cerevisiae.Received 15 January 2004; received after revision 20 February 2004; accepted 4 March 2004     

Evaluation of signal transduction pathways mediating the nuclear exclusion of the androgen receptor by melatonin

The intracellular signaling pathways mediating the nuclear exclusion of the androgen receptor (AR) by melatonin were evaluated in PC3 cells stably transfected with the AR. The melatonin-induced nuclear exclusion of the AR by melatonin (100 nM, 3 h) was blocked by LY 83583 (an inhibitor of guanylyl cyclases). 8-Bromo-cGMP (a cell-permeable cGMP analog), mimicked the effect of melatonin, as did ionomycin (a calcium ionophore) and PMA [an activator of protein kinase C (PKC)], and their effects were blocked by GF-109203X (a selective PKC inhibitor). BAPTA (an intracellular calcium chelator) blocked the effects of melatonin and 8-bromo-cGMP but not of PMA. Inhibition or activation of the protein kinase A pathway did not affect basal or melatonin-mediated AR localization. We conclude that the melatonin-mediated rise in cGMP elicits AR nuclear exclusion via a pathway involving increased intracellular calcium and PKC activation. These results define a novel signaling pathway that regulates AR localization and androgen responses in target cells. Received 31 July 2001; received after revision 18 September 2001; accepted 30 October 2001