Target fishing reveals PfPYK-1 and PfRab6 as potential targets of an antiplasmodial 4-anilino-2-trichloromethylquinazoline hit compound.

We present investigations about the mechanism of action of a previously reported 4-anilino-2-trichloromethylquinazoline antiplasmodial hit-compound (Hit A), which did not share a common mechanism of action with established commercial antimalarials and presented a stage-specific effect on the erythrocytic cycle of P. falciparum at 8 < t < 16 h. The target of Hit A was searched by immobilising the molecule on a solid support via a linker and performing affinity chromatography on a plasmodial lysate. Several anchoring positions of the linker (6,7 and 3') and PEG-type linkers were assessed, to obtain a linked-hit molecule displaying in vitro antiplasmodial activity similar to that of unmodified Hit A. This allowed us to identify the PfPYK-1 kinase and the PfRab6 GTP-ase as potential targets of Hit A.

The transcriptional repressor ICER and cAMP-induced programmed cell death.

The cAMP pathway plays a central role in the response to hormonal signals for cell proliferation, differentiation and apoptosis. In IPC-81 leukaemia cells, activation of the cAMP pathway by prostaglandin E1 treatment, or other cAMP-elevating agents, induces apoptosis within 4-6 h. Inhibition of mRNA or protein synthesis during the first 2 h of cAMP induction protects cells from apoptosis, suggesting a requirement for early gene expression. cAMP-dependent protein kinase phosphorylates a class of nuclear factors and thereby regulates the transcription of a specific set of genes. Here we show that CREM (cAMP Responsive Element Modulator) expression is induced rapidly upon prostaglandin E1 treatment of IPC-81 cells. The induced transcripts correspond to the early product ICER (Inducible cAMP Early Repressor). ICER expression remains elevated until the burst of cell death. Protein synthesis inhibitors which prevent cAMP-induced apoptosis also block de novo ICER synthesis. Transfected IPC-81 cell lines, constitutively expressing high level of ICER are resistant to cAMP-induced cell death. In these transfected cells, cAMP fails to upregulate the ICER transcripts demonstrating that ICER exerts strongly its repressor function on CRE-containing genes. That an early expression of ICER blocks apoptosis, suggests that gene repression by endogenous ICER in IPC-81 is insufficient or occurs too late to protect cells against death. ICER transfected cells rescued from cAMP-induced apoptosis are growth arrested. It shows for the first time that CREM activation directly participates to the decision of the cell to die. ICER, by sequentially repressing distinct sets of CRE-containing genes could modulate cell fate.

Ectopic expression of Bcl-2 switches over nuclear signalling for cAMP-induced apoptosis to granulocytic differentiation.

The IPC-81 myeloid leukaemia cells undergo apoptosis rapidly after cAMP stimulation (6 h) and cell death is prevented by early over-expression of the cAMP-inducible transcription repressor ICER, that blocks cAMP-dependent nuclear signalling. Therefore, the expression of specific genes controlled by CRE-containing promoters is likely to determine cell fate. We now show that cAMP-induced cell death also is abrogated by the over-expression of the anti-apoptotic gene, Bcl-2. Contrary to ICER, Bcl-2 does not affect cAMP-signalling and allows the analysis of cAMP responses in death rescued cells. The Bcl-2 transfected cells treated with 8-CPT-cAMP were growth-arrested and thereafter cells embarked in granulocytic differentiation, with no additional stimulation. Neutrophilic polynuclear granulocytes benefited from a long life span in G0-G1 and remained functional (phagocytosis). This work demonstrates that, using anti-apoptosis regulators, 'death signals' could be exploited to trigger distinct biological responses. Indeed, cAMP signal can trigger several simultaneously developing biological programs, in the same cell, i.e., growth regulation, apoptosis and differentiation. This cell system should prove useful to determine how a tumour cell can be re-programmed for either apoptosis or functional maturation by physiological signals.

Ethylene glycol ethers as hemopoietic toxins--in vitro studies of acute exposure.

Ethylene glycol ethers and their acetate derivatives were analyzed for their toxicity in vitro on several hemopoietic cell lines, either growth-factor-dependent or leukemic, in mouse, rat, and human species. Considering the concentrations that reduced the cell viability in culture by 50%, most of the ethylene glycol ethers and in particular ethylene glycol monoethyl ether (EGEE) or ethylene glycol monobutyl ether (EGBE) should be considered as hemopoietic toxins. EGBE was found to be the most potent toxin on the human promyelocytic cell line, NB4 (median inhibitory concentration (IC50) 5 mM at 6 h; IC50 0.1 mM at 96 h) but also on the factor-dependent cell line DA1 (IC50 80 microM at 48 h). Factor-dependent cell lines were not significantly more sensitive than leukemic cell lines. The toxicity of these compounds falls in the same range of concentration as benzene or phenol, but hydroquinone was significantly more toxic in the same assay (IC50 3-15 microM at 48 h). Toxic effects increased linearly with time. The toxicity of ethylene glycol ethers was confirmed by both assays for colony-forming units in culture medium (CFU-C) (human blood cord cells) and murine bone marrow long-term culture (IC50 5-10 mM). Stromal cells in the adherent layer were more resistant than hemopoietic cells. An all or none toxicity was found within a narrow range of concentration (2-5 mM for EGBE), and chronic exposure over two months did not show cumulative effects on the culture cellularity. The possibility that fibroblastic or macrophage cells worked at the detoxification of the culture is suggested. Results are discussed with regard to epidemiological and in vivo experimental data presently available.

A sustained increase in the endogenous level of cAMP reduces the retinoid concentration required for APL cell maturation to near physiological levels.

cAMP-dependent signal transduction co-operates with retinoids to induce acute promyelocytic leukaemia (APL) cell maturation. The rationale of this work was to determine whether signal cross-talk could be used to decrease the pharmacological doses of retinoids in the treatment of APL. When only the basal level of adenylate-cyclase (AC) activity is present in NB4 cells, up to 1 microM concentration of all-trans retinoic acid (RA) is required for full maturation (100%). In these conditions, with only 10 nM RA less than 20% of cells will differentiate. Although the use of membrane receptor agonists to activate AC has been proved to synergize with RA treatment, these agents were never as potent as cell permeant cAMP analogues. Analogues have disadvantages since cleavage by serum and cellular phosphodiesterases generates metabolites which interfere in cellular response. In the present study, we observed cell maturation by engrafting an autonomous Bordetella pertussis AC which steadily delivers natural cAMP into the cell. The enzyme alone had no effect on cell maturation. Importantly, cell maturation was increased in a dose-dependent manner when the bacterial AC (1 ng/ml to 1 microg/ml) was used to potentiate the effects of low doses RA (10 nM). More than 50% of cells matured with only 10 nM of RA and 200 ng/ml of B. pertussis AC. The maturation response was significantly increased when lower amounts of enzyme were repetitively added to the culture to compensate for enzymatic decay. These results indicate that a sustained AC activity enhanced cell maturation. We were able to reduce to 3 nM the RA requirement, provided that a minimal amount (20 ng/ml) of B. pertussis AC was added every 12 h in culture. Membrane signalling maintaining high the level of cAMP substantially improved the efficacy of APL cell maturation by retinoids. Therefore, therapeutic benefits are expected by lowering the concentration of RA towards physiological (nanomolar) levels, thus reducing the side-effects of the drug. cAMP-elevating drugs that act on a post-cyclase target (cyclic-nucleotide phosphodiesterases) or cell-targeted drug carriers (cAMP and RA loaded liposomes) should be evaluated as maturation therapies combining the activation of multiple signalling pathways.

A retinoid acid 'resistant' t(15;17) acute promyelocytic leukemia cell line: isolation, morphological, immunological, and molecular features.

We report the isolation of a maturation-resistant acute promyelocytic leukemia (APL) cell line. This permanent cell line, derived from the same patient as the maturation inducible NB4 cell line, is the first retinoid-resistant cell line with a t(15;17) chromosomal translocation. Morphological, immunocytochemical and molecular features of the maturation responsive (NB4) and unresponsive (NB4-RAr) cells are compared. The isolation of the NB4-RAr cell line occurred through a two-step process requiring the continuous selective pressure of all-trans-retinoic acid. Cells are also resistant to 13-cis-retinoic acid. Karyotypic and Southern-blot analyses show that the two cell lines are similar with respect to the translocation. Northern-blot analyses show that the chimeric fusion transcript PML-RAR alpha and the normal allelic PML and RAR alpha transcripts are similarly expressed in both cell lines. The molecular basis for unresponsiveness to retinoic acid is not known. This resistant cell line offers a cellular model for molecular biology studies on the mechanism of induction of APL cell maturation, as well as a means to elucidate the molecular mechanisms of resistance. It also furnishes a unique tool for designing and/or screening new therapeutic drugs to avoid or relieve retinoid maturation blockage.

Multiple apoptotic death types triggered through activation of separate pathways by cAMP and inhibitors of protein phosphatases in one (IPC leukemia) cell line.

The protein phosphatase inhibitors okadaic acid and calyculin A at moderate concentrations induced three types of apoptotic promyelocytic leukemia cell death, distinct with respect to ultrastructure and polynucleotide fragmentation. Calyculin A at higher concentrations (> 50 nM) induced a non-apoptotic death type with high ATP and pronounced micromitochondriosis. This suggests that protein phosphorylation pathways are involved in the triggering of several death pathways. Activation of the cAMP kinase induced yet another apoptotic death type, preferentially affecting cells in S-phase. In fact, cAMP acted in two ways to stop IPC promyelocyte proliferation: (1) block in late G1 (preventing new cells from entering DNA replication); and (2) induction of apoptosis in S-phase. cAMP and phosphatase inhibitors acted via distinct pathways. The inhibitors suppressed cAMP-induced death, but only at concentrations high enough to commit the cells to alternative, less conspicuous death types. The tumor-promoting activity of okadaic acid and calyculin A may therefore not be by protection against apoptosis. DNA fragmentation correlated with the novel feature of limited 28 S rRNA cleavage, suggesting co-ordinated polynucleotide cleavage, possibly directed against illegitimate polynucleotides, in some apoptotic death types.

RAR-independent RXR signaling induces t(15;17) leukemia cell maturation.

Although retinoic acid receptor alpha (RARalpha) agonists induce the maturation of t(15;17) acute promyelocytic leukemia (APL) cells, drug treatment also selects leukemic blasts expressing PML-RARalpha fusion proteins with mutated ligand-binding domains that no longer respond to all-trans retinoic acid (ATRA). Here we report a novel RARalpha-independent signaling pathway that induces maturation of both ATRA-sensitive and ATRA-resistant APL NB4 cells, and does not invoke the ligand-induced alteration of PML-RARalpha signaling, stability or compartmentalization. This response involves a cross-talk between RXR agonists and protein kinase A signaling. Our results indicate the existence of a separate RXR-dependent maturation pathway that can be activated in the absence of known ligands for RXR heterodimerization partners.

Two distinctly regulated events, priming and triggering, during retinoid-induced maturation and resistance of NB4 promyelocytic leukemia cell line.

In t(15;17) acute promyelocytic leukemia, all-trans retinoic acid (RA) induces leukemic cell maturation in vitro and remission in acute promyelocytic leukemia patients, but in vivo treatments invariably lead to relapse with resistance to RA. NB4, a maturation-inducible cell line, and NB4-RAr sublines (R1 and R2) displaying no maturation in the presence of RA have been isolated from a patient in relapse. We show that resistance to maturation is not a mere unresponsiveness to RA: rather, R1 "resistant" cells do respond to RA (1 microM) by sustained growth, become competent to undergo terminal maturation, and up-regulate CD11c/CD18 integrins. Interestingly, maturation of "resistant" cells, rendered competent by RA, can be achieved by cAMP-elevating agents (prostaglandin E, isoproterenol, cholera toxin, or phosphodiesterase inhibitor) or stable agonistic cAMP analogs such as (SP)-8-chloroadenosine cyclic 3',5'-phosphorothioate. This shows that activation of cAMP-dependent protein kinase (cA kinase) can override the RA resistance and suggests interdependent RA and cAMP signaling pathways in acute promyelocytic leukemia maturation. No such cooperation was observed in the R2 resistant cells, though their cA-kinase was functional. (RP)-8-Chloroadenosine cyclic 3',5'-phosphorothioate, which by displacing endogenous cAMP inhibits the basal cA-kinase activity, decreased the response of sensitive cells to RA. This raises the possibility that cA-kinase plays a key role in the maturation also of RA-sensitive cells. Our results define two discrete steps in the maturation process: an RA-dependent priming step that maintains proliferation while cells become competent to undergo maturation in response to retinoids and a cAMP-dependent step that triggers RA-primed cells to undergo terminal maturation. Uncoupling RA and cAMP action might cause the so-called "resistance."

A dynamic connection between centromeres and ND10 proteins.

ND10, otherwise known as nuclear dots, PML nuclear bodies or PODs, are punctate foci in interphase nuclei that contain several cellular proteins. The functions of ND10 have not been well defined, but they are sensitive to external stimuli such as stress and virus infection, and they are disrupted in malignant promyelocytic leukaemia cells. Herpes simplex virus type 1 regulatory protein Vmw110 induces the proteasome-dependent degradation of ND10 component proteins PML and Sp100, particularly the species of these proteins which are covalently conjugated to the ubiquitin-like protein SUMO-1. We have recently reported that Vmw110 also induces the degradation of centromere protein CENP-C with consequent disruption of centromere structure. These observations led us to examine whether there were hitherto undetected connections between ND10 and centromeres. In this paper we report that hDaxx and HP1 (which have been shown to interact with CENP-C and Sp100, respectively) are present in a proportion of both ND10 and interphase centromeres. Furthermore, the proteasome inhibitor MG132 induced an association between centromeres and ND10 proteins PML and Sp100 in a significant number of cells in the G(2) phase of the cell cycle. These results imply that there is a dynamic, cell cycle regulated connection between centromeres and ND10 proteins which can be stabilised by inhibition of proteasome-mediated proteolysis.