Frode Selheim

cAMP effector mechanisms. Novel twists for an ‘old’ signaling system

Cyclic AMP (cAMP) has traditionally been thought to act exclusively through cAMP‐dependent protein kinase (cAPK, PKA), but a growing number of cAMP effects are not attributable to general activation of cAPK. At present, cAMP is known also to directly regulate ion channels and the ubiquitous Rap guanine exchange factors Epac 1 and 2. Adding to the sophistication of cAMP signaling is the fact that (1) the cAPK holoenzyme is incompletely dissociated even at saturating cAMP, the level of free R subunit of cAPK being able to regulate the maximal activity of cAPK, (2) cAPK activity can be modulated by oxidative glutathionylation, and (3) cAPK is anchored close to relevant substrates, other signaling enzymes, and local compartments of cAMP. Finally, we will demonstrate an example of fine‐tuning of cAMP signaling through synergistic induction of neurite extensions by cAPK and Epac.

The progenitor cell marker NG2/MPG promotes chemoresistance by activation of integrin-dependent PI3K/Akt signaling

Chemoresistance represents a major problem in the treatment of many malignancies. Overcoming this obstacle will require improved understanding of the mechanisms responsible for this phenomenon. The progenitor cell marker NG2/melanoma proteoglycan (MPG) is aberrantly expressed by various tumors, but its role in cell death signaling and its potential as a therapeutic target are largely unexplored. We have assessed cytotoxic drug-induced cell death in glioblastoma spheroids from 15 patients, as well as in five cancer cell lines that differ with respect to NG2/MPG expression. The tumors were treated with doxorubicin, etoposide, carboplatin, temodal, cisplatin and tumor necrosis factor (TNF)α. High NG2/MPG expression correlated with multidrug resistance mediated by increased activation of α3β1 integrin/PI3K signaling and their downstream targets, promoting cell survival. NG2/MPG knockdown with shRNAs incorporated into lentiviral vectors attenuated β1 integrin signaling revealing potent antitumor effects and further sensitized neoplastic cells to cytotoxic treatment in vitro and in vivo. Thus, as a novel regulator of the antiapoptotic response, NG2/MPG may represent an effective therapeutic target in several cancer subtypes.

Identification of functional VEGF receptors on human platelets

Platelets secrete platelet‐derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) upon stimulation. We have demonstrated that platelets have functionally active PDGF α‐receptors, a transmembrane tyrosine kinase involved in negative feedback regulation. Here we demonstrate the presence of the related VEGF receptors fms‐like tyrosine kinase‐1 and kinase‐insert domain region on human platelets. VEGF itself did not cause platelet aggregation. However, addition of exogenous VEGF to SFRLLN or thrombin‐stimulated platelets potentiated platelet aggregation. Moreover, thrombin‐induced phosphoinositide 3‐kinase and mitogen‐activated protein kinase activity were enhanced in the presence of VEGF.

Microcystin Produces Disparate Effects on Liver Cells in a Dose Dependent Manner

In this review we present recent studies on the effects of the protein phosphatase inhibitor microcystin on mammalian cells. Whereas high concentrations of microcystin promote liver cell death induced by ROS signalling without the involvement of typical apoptotic proteins, intermediate doses activate classic apoptotic pathways. Low concentrations however, increase liver cell survival and proliferation, and can cause primary liver cancer.

Marine Benthic Cyanobacteria Contain Apoptosis-Inducing Activity Synergizing with Daunorubicin to Kill Leukemia Cells, but not Cardiomyocytes

The potential of marine benthic cyanobacteria as a source of anticancer drug candidates was assessed in a screen for induction of cell death (apoptosis) in acute myeloid leukemia (AML) cells. Of the 41 marine cyanobacterial strains screened, more than half contained cell death-inducing activity. Several strains contained activity against AML cells, but not against non-malignant cells like hepatocytes and cardiomyoblasts. The apoptotic cell death induced by the various strains could be distinguished by the role of caspase activation and sensitivity to the recently detected chemotherapy-resistance-associated prosurvival protein LEDGF/p75. One strain (M44) was particularly promising since its activity counteracted the protective effect of LEDGF/p75 overexpressed in AML cells, acted synergistically with the anthracycline anticancer drug daunorubicin in AML cells, and protected cardiomyoblasts against the toxic effect of anthracyclines. We conclude that culturable benthic marine cyanobacteria from temperate environments provide a promising and hitherto underexploited source for novel antileukemic drugs.

Drug repurposing: sulfasalazine sensitizes gliomas to gamma knife radiosurgery by blocking cystine uptake through system Xc(-), leading to glutathione depletion.

Glioblastomas (GBMs) are aggressive brain tumors that always recur after radiotherapy. Cystine, mainly provided by the system Xc− antiporter, is a requirement for glioma cell synthesis of glutathione (GSH) which has a critical role in scavenging free radicals, for example, after radiotherapy. Thus, we hypothesized that the Xc−-inhibitor sulfasalazine (SAS) could potentiate the efficacy of radiotherapy against gliomas. Here, we show that the catalytic subunit of system Xc−, xCT, was uniformly expressed in a panel of 30 human GBM biopsies. SAS treatment significantly reduced cystine uptake and GSH levels, whereas it significantly increased the levels of reactive oxygen species (ROS) in glioma cells in vitro. Furthermore, SAS and radiation synergistically increased DNA double-strand breaks and increased glioma cell death, whereas adding the antioxidant N-acetyl-L-cysteine (NAC) reversed cell death. Moreover, SAS and gamma knife radiosurgery (GKRS) synergistically prolonged survival in nude rats harboring human GBM xenografts, compared with controls or either treatment alone. In conclusion, SAS effectively blocks cystine uptake in glioma cells in vitro, leading to GSH depletion and increased ROS levels, DNA damage and cell death. Moreover, it potentiates the anti-tumor efficacy of GKRS in rats with human GBM xenografts, providing a survival benefit. Thus, SAS may have a role as a radiosensitizer to enhance the efficacy of current radiotherapies for glioma patients.

A Novel Cyanobacterial Nostocyclopeptide is a Potent Antitoxin against Microcystins

Cyanobacterial hepatotoxins (microcystins and nodularins) cause numerous animal poisonings worldwide each year and are threats to human health. However, we found that extracts from several cyanobacteria isolates failed to induce hepatotoxicity even if they contained high concentrations of the liver toxin microcystin. The antitoxic activity abolishes all morphological hallmarks of microcystin‐induced apoptosis, and therefore invalidates cell‐based assays of the microcystin content of bloom‐forming cyanobacteria. The antitoxin was purified from a cyanobacterial isolate (Nostoc sp. XSPORK 13A) from the Baltic Sea, and the activity was shown to reside in a novel cyclic peptide of the nostocyclopeptide family (nostocyclopeptide M1, Ncp‐M1) that consists of seven amino acids (Tyr1‐Tyr2‐D‐HSe3‐L‐Pro4‐L‐Val5‐(2S,4S)‐4‐MPr6‐Tyr7; MW=881) with an imino linkage between Tyr1 and Tyr7. Ncp‐M1 did not compete with labelled microcystin for binding to protein phosphatase 2A; this explains why the antitoxin did not interfere with phosphatase‐based microcystin assays. Currently used agents that interfere with microcystin action, such as inhibitors of ROS formation, microcystin uptake and Cam‐kinase activity, are themselves inherently toxic. Since Ncp‐M1 is potent and nontoxic it promises to become a useful mechanistic tool as soon as its exact cellular target is elucidated.

PI 3-kinase signalling in platelets: the significance of synergistic, autocrine stimulation.

Phosphoinositide 3-kinases (PI 3Ks) play a key role in regulation of intracellular signalling and cellular function, including cell proliferation, apoptosis, chemotaxis, membrane trafficking and platelet activation. The PI 3Ks are grouped into three classes on the basis on their structure and in vitro substrate specificity. Class I are activated by a variety of agonists which mediate their effect through tyrosine kinase-linked or G-proteinlinked receptors. In vivo class I PI 3Ks seem to preferentially phosphorylate the D3 hydroxyls of the inositol moiety of PtdIns(4,5)P 2 to produce PtdIns(3,4,5)P 3 . However, class II PI 3Ks preferentially phosphorylate the D3 hydroxyl of PtdIns and PtdIns(4)P to produce PtdIns(3)P and PtdIns(3,4)P 2 , respectively. The late accumulation of PtdIns(3,4)P 2 has been suggested to play an important role in irreversible platelet aggregation. In human platelets the class II PI 3K isoform HsC2-PI 3K is activated in an integrin alpha Ib beta 3 +fibrinogen dependent manner. Class III PI 3Ks phosphorylate PtdIns to produce PtdIns(3)P, which play a crucial role in vesicular trafficking. Recent work has suggested that crosstalk between individual receptors and their downstream signal pathways play a central role in PI 3K signalling responses. In this review, we will concentrate on recent advances regarding the regulation of platelet PI 3Ks. IPhosphoinositide 3-kinases (PI 3Ks) play a key role in regulation of intracellular signalling and cellular function, including cell proliferation, apoptosis, chemotaxis, membrane trafficking and platelet activation. The PI 3Ks are grouped into three classes on the basis on their structure and in vitro substrate specificity. Class I are activated by a variety of agonists which mediate their effect through tyrosine kinase-linked or G-protein-linked receptors. In vivo class I PI 3Ks seem to preferentially phosphorylate the D3 hydroxyls of the inositol moiety of PtdIns(4,5)P2 to produce PtdIns(3,4,5)P3. However, class II PI 3Ks preferentially phosphorylate the D3 hydroxyl of PtdIns and PtdIns(4)P to produce PtdIns(3)P and PtdIns(3,4)P2, respectively. The late accumulation of PtdIns(3,4)P2 has been suggested to play an important role in irreversible platelet aggregation. In human platelets the class II PI 3K isoform HsC2-PI 3K is activated in an integrin alpha IIb beta 3 + fibrinogen-dependent manner. Class III PI 3Ks phosphorylate PtdIns to produce PtdIns(3)P, which play a crucial role in vesicular trafficking. Recent work has suggested that crosstalk between individual receptors and their downstream signal pathways play a central role in PI 3K signalling responses. In this review, we will concentrate on recent advances regarding the regulation of platelet PI 3Ks.

Performance of super‐SILAC based quantitative proteomics for comparison of different acute myeloid leukemia (AML) cell lines

As a direct consequence of the high diversity of the aggressive blood cancer acute myeloid leukemia (AML), proteomic samples from patients are strongly heterogeneous, rendering their accurate relative quantification challenging. In the present study, we investigated the benefits of using a super‐SILAC mix of AML derived cell lines as internal standard (IS) for quantitative shotgun studies. The Molm‐13, NB4, MV4‐11, THP‐1, and OCI‐AML3 cell lines were selected for their complementarity with regard to clinical, cytogenetic, and molecular risk factors used for prognostication of AML patients. The resulting IS presents a high coverage of the AML proteome compared to single cell lines allied with high technical reproducibility, thus enabling its use for AML patient comparison. This was confirmed by comparing the protein regulation between the five cell lines and by applying the IS to patient material; hence, we were able to reproduce specific functional regulations known to be related to disease progression and molecular genetic abnormalities. The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000441.

Chromosome 19 Annotations with Disease Speciation: A First Report from the Global Research Consortium

A first research development progress report of the Chromosome 19 Consortium with members from Sweden, Norway, Spain, United States, China and India, a part of the Chromosome-centric Human Proteome Project (C-HPP) global initiative, is presented ( http://www.c-hpp.org ). From the chromosome 19 peptide-targeted library constituting 6159 peptides, a pilot study was conducted using a subset with 125 isotope-labeled peptides. We applied an annotation strategy with triple quadrupole, ESI-Qtrap, and MALDI mass spectrometry platforms, comparing the quality of data within and in between these instrumental set-ups. LC-MS conditions were outlined by multiplex assay developments, followed by MRM assay developments. SRM was applied to biobank samples, quantifying kallikrein 3 (prostate specific antigen) in plasma from prostate cancer patients. The antibody production has been initiated for more than 1200 genes from the entire chromosome 19, and the progress developments are presented. We developed a dedicated transcript microarray to serve as the mRNA identifier by screening cancer cell lines. NAPPA protein arrays were built to align with the transcript data with the Chromosome 19 NAPPA chip, dedicated to 90 proteins, as the first development delivery. We have introduced an IT-infrastructure utilizing a LIMS system that serves as the key interface for the research teams to share and explore data generated within the project. The cross-site data repository will form the basis for sample processing, including biological samples as well as patient samples from national Biobanks.