André Sulen

Increased antileukemic effects in human acute myeloid leukemia by combining HSP70 and HSP90 inhibitors

Background: Heat shock proteins (HSPs) are molecular chaperones that assist proteins in their folding to native structures. HSP90, and more recently HSP70, have emerged as possible therapeutic targets in human malignancies, including acute myeloid leukemia (AML). Design and methods: The authors investigated the effects of the HSP70 inhibitor VER-155008 tested alone or in combination with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) on proliferation, viability, constitutive cytokine release and intracellular HSP levels of primary human AML cells. Results: VER-155008 caused a dose-dependent inhibition of cytokine-dependent AML cell proliferation both in suspension cultures and in a colony formation assay, and the drug also had a proapoptotic effect. HSP70 and HSP90 inhibition had additive antiproliferative and proapoptotic effects. VER-155008 caused a strong inhibition of the constitutive AML cell release of several growth factors/regulators of hematopoiesis (i.e., TNF-α, VEGF, IL-3, IL-1β, IL-1 receptor antagonist), but had relatively weak effects on the constitutive chemokine release. HSP70 inhibition did not induce any compensatory increase of other HSPs. Conclusion: HSP70 inhibition has antileukemic effects when tested alone, and the combination of HSP70 and HSP90 inhibition seems to have additive antileukemic effects for primary human AML cells in vitro.

Expression of TP53 Isoforms p53β or p53γ Enhances Chemosensitivity in TP53null Cell Lines

The carboxy-terminal truncated p53 alternative spliced isoforms, p53β and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53null background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53β and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53β and p53γ congenic H1299 was accompanied by increased p21(CIP1/WAF1), Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53β protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level.

Heat shock protein 70 – the next chaperone to target in the treatment of human acute myelogenous leukemia?

Introduction: Heat shock proteins (HSPs) are molecular chaperones that stabilize folding and conformation of mature proteins. HSPs are, therefore, considered as possible therapeutic targets in the treatment of human cancers, including acute myeloid leukemia (AML). This strategy offers the possibility of targeting several oncogenic proteins or several intracellular signaling pathways through the use of a single therapeutic agent. Areas covered: Several specific HSP70 inhibitors have now been developed. We describe: i) the molecular structure of HSP70 and the molecular interactions especially with its co-chaperones; ii) the immunoregulatory functions of HSP70; iii) the expression and function of HSP70 in human AML cells; iv) the molecular and pharmacological characteristics of potential HSP70 inhibitors; and v) the clinical and experimental studies of HSP70 inhibition in human AML. Our review is based on careful selection of relevant publications identified in the PubMed database. Expert opinion: HSP70 inhibitors have antileukemic activity in human AML. However, additional experimental studies using in vitro models as well as animal models are required, including detailed toxicology studies, as a scientific basis for the optimal design of future clinical studies.

Gel2DE - A software tool for correlation analysis of 2D gel electrophoresis data

BackgroundTwo-dimensional gel electrophoresis (2DE) is a powerful technique for studying protein isoforms and their modifications. Existing commercial 2D image analysis tools rely on spot detection that limits analysis of complex protein profiles, e.g. spot appearance/disappearance or overlapping spots. Pixel-by-pixel correlation analysis, an analysis technique for identifying relations between protein patterns in gel images and external variables, can overcome such limitations in spot analysis.ResultsWe have implemented the first publically available pixel-by-pixel correlation analysis tool, the software Gel2DE. 2D immunoblot time course analysis of p53 protein stabilization in response to ionizing irradiation shows that pixel-by-pixel analysis can yield an overall activation biosignature for p53, despite changing spots shape, size and position.ConclusionsPixel-by-pixel correlation of aligned 2D images permits analysis of complex protein patterns. We anticipate that the Gel2DE correlation software will be a useful tool for future bioinformatics discoveries through 2D gel electrophoresis.

Distinct single cell signal transduction signatures in leukocyte subsets stimulated with khat extract, amphetamine-like cathinone, cathine or norephedrine

BackgroundAmphetamine and amphetamine derivatives are suggested to induce an immunosuppressive effect. However, knowledge of how amphetamines modulate intracellular signaling pathways in cells of the immune system is limited. We have studied phosphorylation of signal transduction proteins (Akt, CREB, ERK1/2, NF-κB, c-Cbl, STAT1/3/5/6) and stress sensors (p38 MAPK, p53) in human leukocyte subsets following in vitro treatment with the natural amphetamine cathinone, the cathinone derivatives cathine and norephedrine, in comparison with a defined extract of the psychostimulating herb khat (Catha edulis Forsk.). Intracellular protein modifications in single cells were studied using immunostaining and flow cytometry, cell viability was determined by Annexin V-FITC/Propidium Iodide staining, and T-lymphocyte proliferation was measured by 3H-thymidine incorporation.ResultsCathinone, cathine and norephedrine generally reduced post-translational modifications of intracellular signal transducers in T-lymphocytes, B-lymphocytes, natural killer cells and monocytes, most prominently affecting c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), p38 MAPK (p-Thr180/p-Tyr182) and p53 (both total p53 protein and p-Ser15). In contrast, the botanical khat-extract induced protein phosphorylation of STAT1 (p-Tyr701), STAT6 (p-Tyr641), c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), NF-κB (p-Ser529), Akt (p-Ser473), p38 MAPK (p-Thr180/p-Tyr182), p53 (Ser15) as well as total p53 protein. Cathinone, cathine and norephedrine resulted in unique signaling profiles, with B-lymphocytes and natural killer cells more responsive compared to T-lymphocytes and monocytes. Treatment with norephedrine resulted in significantly increased T-lymphocyte proliferation, whereas khat-extract reduced proliferation and induced cell death.ConclusionsSingle-cell signal transduction analyses of leukocytes distinctively discriminated between stimulation with cathinone and the structurally similar derivatives cathine and norephedrine. Cathinone, cathine and norephedrine reduced phosphorylation of c-Cbl, ERK1/2, p38 MAPK and p53(Ser15), and norephedrine induced T-lymphocyte proliferation. Khat-extract induced protein phosphorylation of signal transducers, p38 MAPK and p53, followed by reduced cell proliferation and cell death. This study suggests that protein modification-specific single-cell analysis of immune cells could unravel pharmacologic effects of amphetamines and amphetamine-like agents, and further could represent a valuable tool in elucidation of mechanism(s) of action of complex botanical extracts.

Leukocyte p53 protein biosignature through standard-aligned two-dimensional immunoblotting ☆

Peripheral leukocytes may reflect systemic disease and stress states through their gene expression profile. Subsequent protein analyses of leukocytes are hypothesized to provide essential information regarding systemic diseases. We have developed a protein biosignature analysis of the tumour suppressor and cell stress sensor p53 based on two-dimensional gel electrophoresis and immunoblotting, and utilize fluorescently labelled reference standards to significantly improve the alignment and comparison of biosignatures, including full-length p53 and isoforms p53β and p53γ. Analysis of the p53 biosignatures of peripheral blood mononuclear cells from 526 healthy individuals and 65 acute myeloid leukaemia patients indicated a novel putative p53 protein variant in a subset of individuals (227 of 526 healthy tested). The p53 variant was more distinct in the reference standard aligned biosignatures of healthy individuals, compared to the non-standard aligned leukaemia biosignatures. This approximately 2 kDa heavier variant of p53 appeared with similar frequency in leukemic and healthy test persons, without coinciding with known splice forms or post-translational modifications of p53. We propose that a standardized leukocyte protein biosignature of p53 provides a powerful research tool and indicate how p53 protein biosignatures may be used in future diagnostics. This article is part of a Special Issue entitled: Integrated omics.

Anti-proliferative activity of the NPM1 interacting natural product avrainvillamide in acute myeloid leukemia

Mutated nucleophosmin 1 (NPM1) acts as a proto-oncogene and is present in ~30% of patients with acute myeloid leukemia (AML). Here we examined the in vitro and in vivo anti-leukemic activity of the NPM1 and chromosome region maintenance 1 homolog (CRM1) interacting natural product avrainvillamide (AVA) and a fully syntetic AVA analog. The NPM1-mutated cell line OCI-AML3 and normal karyotype primary AML cells with NPM1 mutations were significantly more sensitive towards AVA than cells expressing wild-type (wt) NPM1. Furthermore, the presence of wt p53 sensitized cells toward AVA. Cells exhibiting fms-like tyrosine kinase 3 (FLT3) internal tandem duplication mutations also displayed a trend toward increased sensitivity to AVA. AVA treatment induced nuclear retention of the NPM1 mutant protein (NPMc+) in OCI-AML3 cells and primary AML cells, caused proteasomal degradation of NPMc+ and the nuclear export factor CRM1 and downregulated wt FLT3 protein. In addition, both AVA and its analog induced differentiation of OCI-AML3 cells together with an increased phagocytotic activity and oxidative burst potential. Finally, the AVA analog displayed anti-proliferative activity against subcutaneous xenografted HCT-116 and OCI-AML3 cells in mice. Our results demonstrate that AVA displays enhanced potency against defined subsets of AML cells, suggesting that therapeutic intervention employing AVA or related compounds may be feasible.

Signaling effects of sodium hydrosulfide in healthy donor peripheral blood mononuclear cells.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter in human physiology and inflammatory disease, however, with limited knowledge of how signal transduction pathways are involved in immune cells. To examine the effects of sulfide on relevant intracellular signaling in human peripheral blood mononuclear cells (PBMCs), we stimulated healthy donor PBMCs with sodium hydrosulfide (NaHS, 1-1000μM) to mimic H2S stimulation, and analyzed phosphorylation of p38 mitogen activated protein kinase (MAPK) (pT180/pY182), NF-κB p65 (pS529), Akt (pS473) and CREB/ATF1 (pS133/pS63) with flow and mass cytometry. In contrast to transient effects in subsets of lymphocytes, classical monocytes demonstrated sustained phosphorylation of p38, Akt and CREB/ATF1. NaHS induced calcium dependent phosphorylation of p38, Akt and CREB, but not NF-κB, and the phosphorylation of Akt was partly dependent on p38, indicative of p38-Akt crosstalk. Attenuation of these effects by molecules targeting p38 and Hsp90 indicated Hsp90 as a possible target for H2S-induced activation of p38. These results provide a description of a NaHS-induced signal transduction pathway in human primary immune cells that may have relevance for the role of sulfides in inflammation.

Evaluation of the effects of clinical diagnostic ultrasound in combination with ultrasound contrast agents on cell stress: Single cell analysis of intracellular phospho-signaling pathways in blood cancer cells and normal blood leukocytes

Clinical diagnostic ultrasound has been known as one of the safest imaging modalities available, yet very little is known about the cellular response to such acoustic conditions. With the increased interest in therapeutic ultrasound it is becoming ever more important to understand the effects of ultrasound on cells.In our work here we investigate the effect of clinical diagnostic ultrasound on several cell signalling proteins (p38 p-Thr180/p-Tyr182, ERK 1/2 p-Thr202/p-Tyr204 and p53 ac-Lys382) on leukaemia cells (MOLM-13) and monocytes. Our results show that leukaemia cells and monocytes react differently to ultrasound and microbubbles. A relatively small increase in p38 signalling was seen in the leukemic cells, and only at higher intensities in combination with microbubbles. In contrast the monocytes showed an increase in p38 signalling at all acoustic intensities with microbubbles and at the high acoustic intensity without microbubbles. Furthermore, the leukemic cells showed an overall increase in ERK 1/2 signalling whereas the monocytes showed a decrease. These results indicate that the leukaemia cells are less sensitive to stress induced by ultrasound and microbubbles when compared to normal monocytes. In conclusion, our results show that clinical diagnostic ultrasound does have a measurable effect on intracellular signalling but may differ drastically between different cell types. This may affect the conditions necessary for therapeutic ultrasound.

Elevated monocyte phosphorylated p38 in nearby employees after a chemical explosion

Personalised health surveillance is infrequent or absent in occupational and environmental medicine. The shortage of functional tests in relevant cells and tissues greatly limits our understanding of environmental exposures and associated disease risk. We evaluated single cell signalling in peripheral blood mononuclear cells from 301 individuals in a cross sectional health survey 18 months after a chemical explosion of sulphorous coker gasoline. The accident created a malodourous environment leading to long-term health complaints. Multiple regression analysis revealed T-cell specific elevated phosphorylation of the stress kinase p-p38 (T180/Y182) among tobacco smokers and monocyte-specific elevated phosphorylation in employees at the explosion site. Other studies of the accident reported reduced tear film stability, and more airway obstruction and subjective health complaints among the employees at the accident site. Elevated monocyte p-p38 in the employee group was independent of such health effects, and could therefore be dependent on the sulphuric malodorous environment. The present study proposes signalling status in leukocytes as a scalable biomarker providing information about environmental exposures.