Øystein Bruserud

Single Cell Profiling of Potentiated Phospho-Protein Networks in Cancer Cells

Altered growth factor responses in phospho-protein-driven signaling networks are crucial to cancer cell survival and pathology. Profiles of cancer cell signaling networks might therefore identify mechanisms by which such cells interpret environmental cues for continued growth. Using multiparameter flow cytometry, we monitored phospho-protein responses to environmental cues in acute myeloid leukemia at the single cell level. By exposing cancer cell signaling networks to potentiating inputs, rather than relying upon the basal levels of protein phosphorylation alone, we could discern unique cancer network profiles that correlated with genetics and disease outcome. Strikingly, individual cancers manifested multiple cell subsets with unique network profiles, reflecting cancer heterogeneity at the level of signaling response. The results revealed a dramatic remodeling of signaling networks in cancer cells. Thus, single cell measurements of phospho-protein responses reveal shifts in signaling potential of a phospho-protein network, allowing for categorizing of cell network phenotypes by multidimensional molecular profiles of signaling.

Histone Deacetylase Inhibitors in Cancer Treatment: A Review of the Clinical Toxicity and the Modulation of Gene Expression in Cancer Cells

Characterization of epigenetic events in carcinogenesis has led to the discovery of a new class of oncogenes and thereby a new class of therapeutic targets. Among the new therapeutic approaches are modulation of protein lysine acetylation through inhibition of histone deacetylases (HDACs). HDACs deacetylate histones as well as transcription factors and can modulate gene expression through both these mechanisms in normal and malignant cells. Furthermore, acetylation is an important posttranslational modulation of several proteins involved in the regulation of cell proliferation, differentiation and apoptosis in normal as well as cancer cells. Even though several HDAC inhibitors have been characterized in vitro, only a limited number of these agents are in clinical trials. Various HDAC inhibitors differ in their toxicity profile when comparing the side effects described in the available clinical studies of HDAC inhibition in the treatment of cancer. These drugs may also affect normal hematopoiesis; hematologic toxicity is common to many drugs but stimulation of hematopoiesis seems to occur for others. HDAC inhibitors usually affect < 10% of the genes in cancer cells. Divergent effects of HDAC inhibition on the global gene expression profiles have been described when testing various cancer cells, and this is further complicated by altered HDAC expression induced by HDAC inhibitors. However, increased p21 expression seems to be a common characteristic for most studies, suggesting an important role of this molecule during HDAC inhibitory treatment. Even though the initial studies are encouraging, additional in vitro and in vivo pharmacological characterization is definitely needed.

The proteasome inhibitors bortezomib and PR-171 have antiproliferative and proapoptotic effects on primary human acute myeloid leukaemia cells.

Proteasome inhibitors represent a new class of antineoplastic drugs that are considered in the treatment of haematological malignancies. We compared the effects of the reversible proteasome inhibitor bortezomib (Velcade®) and the epoxomicin derivative PR‐171, an irreversible inhibitor, on primary human acute myeloid leukaemia (AML) cells. Both drugs inhibited autocrine‐ and cytokine‐dependent proliferation of primary AML blasts when tested at nanomolar levels (0·1–100 nmol/l). The antiproliferative effect was independent of basal chymotrypsin‐like proteasome activity (showing a 20‐fold variation between patients), genetic abnormalities, morphological differentiation and CD34 expression when testing a large group of consecutive patients (n = 54). The effect was retained in cocultures with bone marrow stromal cells. In addition, both drugs enhanced apoptosis. The effect of PR‐171 could be detected at lower concentrations than for bortezomib, especially when testing the influence on clonogenic AML cell proliferation. Both drugs had divergent effects on AML cells’ constitutive cytokine release. Furthermore, both drugs caused a decrease in proliferation and viability when tested in combination with idarubicin or cytarabine. An antiproliferative effect on primary human acute lymphoblastic leukaemia cells was also detected. We conclude that nanomolar levels of the proteasome inhibitors tested had dose‐dependent antiproliferative and proapoptotic effects on primary AML cells in vitro.

Cryopreserving human peripheral blood progenitor cells with 5‐percent rather than 10‐percent DMSO results in less apoptosis and necrosis in CD34+ cells

BACKGROUND : The grade of toxicity experienced by patients when cryopreserved peripheral blood progenitor cells (PBPCs) are reinfused is related to the amount of DMSO present in the PBPC concentrate. This study was initiated to investigate whether cell viability, apoptosis, and necrosis would be altered in CD34+ cells if PBPCs were cryopreserved with 5‐percent as opposed to the conventional 10‐percent DMSO.

A phase II trial of pegylated interferon alpha-2b therapy for polycythemia vera and essential thrombocythemia: feasibility, clinical and biologic effects, and impact on quality of life.

Conventional interferon‐α (IFN) is an effective treatment for patients with myeloproliferative disorders. However, many patients discontinue therapy because of side effects.

In vitro evaluation of metabolic changes and residual platelet responsiveness in photochemical treated and gamma-irradiated single-donor platelet concentrates during long-term storage.

BACKGROUND: Photochemical treatment (PCT) prevents replication of pathogens in platelet concentrates (PCs) by cross‐linking nucleic acids and thus affects all cells containing DNA or RNA.

New Strategies for the Treatment of Acute Myelogenous Leukemia: Differentiation Induction—Present Use and Future Possibilities

A differentiation block and an accumulation of immature myeloid cells characterize acute myelogenous leukemia (AML). However, native AML cells usually show some morphological signs of differentiation that allow a classification into different subsets, and further differentiation may be induced by exposure to various soluble mediators, for example, all‐trans retinoic acid (ATRA) and several cytokines. Combination therapy with ATRA and chemotherapy should now be regarded as the standard treatment of the acute promyelocytic leukemia (APL) variant of AML. Although several agents can also induce leukemic cell differentiation for other AML subgroups, in vitro studies as well as clinical data have demonstrated that these agents often have heterogeneous effects on the leukemic progenitors. This makes the clinical impact of differentiation induction therapy for individual patients difficult to predict. However, differentiation induction should be regarded as a promising therapeutic approach, especially as a part of immunotherapy or in combination with intensive chemotherapy to increase the susceptibility of AML blasts to drug‐induced apoptosis. Although the morphology‐based French‐American‐British classification was used to identify APL as an AML subset that required a special treatment, it seems unlikely that this classification alone can be used to identify new subsets of AML patients with special therapeutic requirements. Future studies on differentiation induction in AML should therefore focus on A) the identification of therapeutic agents with more predictable effects; B) the use of clinical and laboratory parameters to define new subsets of AML patients in which differentiation induction has a predictable and beneficial effect, and C) the characterization of how AML blast sensitivity to drug‐induced apoptosis is altered by differentiation induction.

Cytokine accumulation in photochemically treated and gamma-irradiated platelet concentrates during storage

BACKGROUND:  Photochemical treatment (PCT) for pathogen reduction of platelet concentrates (PCs) affects all cells containing DNA and/or RNA. Soluble mediators, which may cause transfusion reactions, are determined by the balance between secretion and/or cell destruction and binding and/or degradation.

The Crosstalk Between the Matrix Metalloprotease System and the Chemokine Network in Acute Myeloid Leukemia

Matrix metalloproteinases (MMPs) comprise a large family of zinc-dependent endopeptidases, which are best known for their ability to degrade essentially all components of the extracellular matrix (ECM). By breaking down ECM, MMPs may remove physical barriers, thus allowing cells to migrate and potentially invade other tissues. Recent evidence, however, shows that the proteolytic activities of MMPs also affect several fundamental physiological processes. Primary human acute myeloid leukemia (AML) cells often show constitutive release of several MMPs and chemokines, and there seems to be a crosstalk between the MMP system and the chemokine network. Firstly, the nuclear factor-κB (NF-κB) system represents a common regulator at the transcriptional level both for MMPs (e.g. MMP-1 and MMP-9) and for the constitutive release of several chemokines (CCL2-4/CXCL1/8) by primary human AML cells. Secondly, the crosstalk at the molecular level probably includes MMP-mediated structural alteration and activation of constitutively released chemokines involved in AML cell migration (e.g. CXCL12) and stimulation of bone marrow angiogenesis (e.g. CXCL8). Thirdly, at a functional level the two systems interact because the chemokine network plays a role in similar physiological processes as the MMPs, including AML cell proliferation and migration and local regulation of angiogenesis. Both the chemokine system and MMPs are currently being evaluated as targets in anti-angiogenesis/cancer therapy and may also have potential therapeutic implications in AML. This review introduces the different members of the MMP family and describes their interactions with the chemokine network and the possible involvement of MMPs together with chemokines in leukemogenesis and chemosensitivity in AML.

Microvascular endothelial cells increase proliferation and inhibit apoptosis of native human acute myelogenous leukemia blasts

Interactions between acute myelogenous leukemia (AML) blasts and neighbouring endothelial cells in the bone marrow seem important both for disease development and susceptibility to chemotherapy. We investigated the effects of soluble mediators released by microvascular endothelial cells on native human AML cells. AML cells derived from 33 patients were cocultured with microvascular endothelial cells, separated by a semipermeable membrane. We investigated the effect of coculture on AML cell proliferation, viability/apoptosis and cytokine release. Coculture increased AML cell proliferation, and this growth enhancement included the clonogenic leukemia cell subset. Increased release of several soluble mediators was also detected (interleukin 3, interleukin 6, granulocyte‐macrophage and granulocyte colony‐stimulating factors) in cocultures. Our cytokine neutralization experiments suggest that an intercellular crosstalk involving several soluble mediators contribute to the increased leukemia cell proliferation. The presence of endothelial cells had an additional antiapoptotic effect on the AML cells. The endothelial cells did not have any growth‐enhancing effect on native human acute lymphoblastic leukemia cells. Our in vitro results suggest that the release of soluble mediators by microvascular endothelial cells supports leukemic hematopoiesis through paracrine mechanisms by direct enhancement of AML blast proliferation and by inhibition of leukemic cell apoptosis.