Harald Biersack

Cytotoxicity of anthracyclines: correlation with cellular uptake, intracellular distribution and DNA binding.

SummaryIn order to interact with topoisomerase II and induce genotoxicity, anthracyclines have to cross the outer cell membrane and the cytoplasm, enter the nucleus, and bind to the DNA. We incubated sensitive and resistant hematopoietic cells from cell lines and patient cells with daunorubicin, idarubicin, and its active derivative idarubicinol, extracted the anthracyclines from whole cells and nuclei, and determined their concentration fluorimetrically. Additionally, the DNA binding of the drugs was evaluated in the same cells by determining fluorescence resonance energy transfer between the anthracyclines and DNA-bound Hoechst dye 33342. We found a several thousand-fold accumulation of anthracyclines in sensitive and resistant hematopoietic cells; 30–60% of the drugs are found in the nucleus, resulting in 200- to 300-fold differences in concentration between the nucleus and outer fluids. A small proportion of the intracellular or intranuclear anthracyclines is bound to the DNA. The amount of DNA-bound anthracyclines correlates directly to cell death. It takes an additional 10 min for idarubicin and 30 min for daunorubicin to satisfy DNA binding sites after the drugs have arrived in the nucleus. The described methods provide the means to perform ex vivo studies on clinical material.

Prevalence of targetable oncogenic mutations and genomic alterations in Epstein–Barr virus-associated diffuse large B-cell lymphoma of the elderly

Abstract Epstein–Barr virus (EBV)-associated diffuse large B-cell lymphoma (DLBCL) of the elderly constitutes a provisional clinicopathological entity in the current World Health Organization (WHO) classification and its genomic features remain sparsely characterized. We investigated a cohort of 26 cases of untreated de novo EBV-positive DLBCL of the elderly by high-resolution array-based comparative genomic profiling and fluorescence in situ hybridization (FISH). Moreover, we screened for activating mutations affecting nuclear factor (NF)-κB pathway signaling and chromatin remodeling (EZH2, CD79B, CARD11 and MYD88) due to their impact of gene expression signatures and postulated upcoming therapeutic targetability. We identified an overlap between genomic aberrations previously described to be exclusive features of plasmablastic lymphoma (PL), post-transplant lymphoproliferative disorder (PTLD) and DLBCL, respectively, indicating a close cytogenetic relationship between these entities. Few mutations affecting CD79B and CARD11 and no MYD88 mutations were detectable, hinting at EBV-mediated activation of NF-κB as an alternative to pathologically enforced B-cell receptor signaling in this rare entity.

Dasatinib blocks transcriptional and promigratory responses to transforming growth factor-beta in pancreatic adenocarcinoma cells through inhibition of Smad signalling: implications for in vivo mode of action

BackgroundWe have previously shown in pancreatic ductal adenocarcinoma (PDAC) cells that the SRC inhibitors PP2 and PP1 effectively inhibited TGF-β1-mediated cellular responses by blocking the kinase function of the TGF-β type I receptor ALK5 rather than SRC. Here, we investigated the ability of the clinically utilised SRC/ABL inhibitor dasatinib to mimic the PP2/PP1 effect.MethodsThe effect of dasatinib on TGF-β1-dependent Smad2/3 phosphorylation, general transcriptional activity, gene expression, cell motility, and the generation of tumour stem cells was measured in Panc-1 and Colo-357 cells using immunoblotting, reporter gene assays, RT-PCR, impedance-based real-time measurement of cell migration, and colony formation assays, respectively.ResultsIn both PDAC cell lines, dasatinib effectively blocked TGF-β1-induced Smad phosphorylation, activity of 3TPlux and pCAGA(12)-luc reporter genes, cell migration, and expression of individual TGF-β1 target genes associated with epithelial-mesenchymal transition and invasion. Moreover, dasatinib strongly interfered with the TGF-β1-induced generation of tumour stem cells as demonstrated by gene expression analysis and single cell colony formation. Dasatinib also inhibited the high constitutive migratory activity conferred on Panc-1 cells by ectopic expression of kinase-active ALK5.ConclusionsOur data suggest that the clinical efficiency of dasatinib may in part be due to cross-inhibition of tumour-promoting TGF-β signalling. Dasatinib may be useful as a dual TGF-β/SRC inhibitor in experimental and clinical therapeutics to prevent metastatic spread in late-stage PDAC and other tumours.

Activation of topoisomerase II during partial purification by heparin-Sepharose chromatography

Partial purification of topoisomerase II from small samples (10(7)-10(8) cells) of human leukaemic cells was achieved by isolation of cell nuclei, hyper-osmotic extraction of nuclear proteins, sorption of nuclear proteins by heparin-Sepharose and elution with potassium phosphate. Similar results were obtained by gradient and batchwise elution. The calatylic activity of topoisomerase increased ca. eightfold after removal of ca. 95% of the contaminating nuclear proteins. The conserved enzymatic activity after partial purification indicates that the enzyme was not damaged. The half-life of enzymatic activity is increased by the chromatographic procedure. Owing to its high yield and technical simplicity, this could be a candidate procedure for the study of topoisomerase II in patient-derived blood samples.

Nuclear Topoisomerase II Activity Changes During HL-60 Leukemic Cell Differentiation: Alterations in Drug Sensitivity and pH Dependency

We have studied the effect of dimethyl-sulfoxide(DMSO)-induced granulocytic differentiation on the sensitivity of HL-60 cells to various cytotoxic topoisomerase II inhibitors: (i) undifferentiated HL-60 cells are highly sensitive to etoposide, while differentiated HL-60 cells are 700-1000 fold resistant to etoposide, (ii) undifferentiated HL-60 are 50-100 fold resistant against 4-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA) when compared to the differentiated HL-60 cells, (iii) the addition of mAMSA to the medium inhibits granulocytic differentiation of HL-60 cells. This change in resistance pattern is probably due to an alteration of topoisomerases since distinctive iso-activites of topoisomerase can be discriminated on the basis of the pH profile, which alters markedly during differentiation. In an etoposide-resistant HL-60 cell line we found a reduced topoisomerase activity at pH 7.8/7.9. This topoisomerase iso-activity is obviously involved in etoposide cytotoxicity.

Use of anion-exchange chromatography and chromatofocusing to reveal the structural and functional heterogeneity of topoisomerase II in a HL-60 cell line resistant to multi-drug treatment

Fractionation of nuclear extracts from a multi-drug-resistant subclone of the human promyelocytic subline HL-60 by anion-exchange chromatography and chromatofocusing resolves at least two different subtypes of topoisomerase II, which are not identical to the known alpha- and beta-forms of the enzyme because both forms are contained in each subtype. The two subtypes are present in about equal proportions and differ remarkably with respect to the optimum of reaction and sensitivity to m-amsacrine and orthovanadate. Both subtypes are highly insensitive to etoposide inhibition in vitro.

Drug-Sensitivity and DNA-Binding of a Subform of Topoisomerase Iiα in Resistant Human HL-60 Cells

Topoisomerase II alpha (170 kDa) expressed in human HL-60 cells is heterogeneous in charge. By two-dimensional electrophoresis and chromatofocussing two major subforms with pI of 6.5 and 6.7 can be resolved. By preparative anion-exchange chromatography we separated the known topoisomerase II isoenzymes (170/180 kDa) and in addition a late-eluting 170 kDa form, which has not been described before. The catalytic optimum of this late-eluting form is shifted to pH 9.4. It is more than 100-fold resistant to orthovanadate, amsacrine or etoposide, and has an increased salt stability. SDS-treatment induces covalent attachment of this enzyme fraction to calf thymus DNA in the absence of drug. The latter observations indicate an increase in DNA-binding. In the tightly DNA-bound state the late-eluting enzyme is not targeted by cleavable complex forming drugs. Accordingly, cells may become drug-resistant by expressing this form predominantly.

Favorable prognostic impact of RAS mutation status in multiple myeloma treated with high-dose melphalan and autologous stem cell support in the era of novel agents: a single center perspective

Oncogenic mutations affecting members of the RAS gene family (NRAS, KRAS, HRAS) have been recurrently described in many solid tumors and hematological malignancies including multiple myeloma in a variety of frequencies showing entity-dependent patterns of distribution [1]. In the context of multiple myeloma, there appear to be high and approximately equal rates (10–20%) of KRAS and NRAS mutations with a significant predominance over mutations affecting other members of the RAS gene family [2]. While RAS mutant myeloma cells are traditionally considered to be highly refractory to establish treatment approaches including reduced cytotoxicity of nitrogen mustard alkylating agent melphalan, recent in vitro assays revealed a dominant addiction of RAS mutants to an operational proteasome [3,4]. Moreover, no in vivo data on the association of RAS mutations with chemoresistance towards melphalan have been reported so far. A recently published randomized trial comparing chemotherapy plus lenalidomide with autologous stem cell transplantation (ASCT) followed by maintenance with lenalidomide-prednisone or lenalidomide alone in patients with newly diagnosed myeloma demonstrated significantly prolonged progression-free survival (PFS) in the transplant arm (60% vs 38% at 3 years), thus widely settling the question whether ASCT still has a role in myeloma treatment in the age of novel agents [5]. Novel agent-based (e.g. thalidomide, lenalidomide and bortezomib) combination therapies have emerged as the contemporary standard of care for induction therapy prior to ASCT in multiple myeloma; moreover, they represent viable options for treatment at relapse, maintenance therapy or in transplant ineligible patients [5–7]. Contemporary transplant trials involving novel agents for induction therapy revealed that responses could be deepened to the effect that the number of patients achieving complete response (CR) and very good partial response (VGPR) is increased between induction and the posttransplant consolidation [8]. Investigations pre-dating the introduction of novel agents reported especially KRAS mutations to be associated with unfavorable prognosis, whilst recent studies focusing on bortezomib-based treatment for relapsed and refractory myeloma identified NRAS rather then KRAS mutations to herald inferior response in refractory and relapsed multiple myeloma [2,9,10]. Pathogenetic, prognostic and therapeutic implications of RAS mutations in multiple myeloma constitute a widely unresolved matter and no profound consideration has been given to the role of ASCT in this context, especially in the era of proteasome inhibition and immunomodulatory treatment. To our knowledge, this retrospective study is the first assessment of the prognostic impact of RAS mutations on clinical outcome of multiple myeloma patients treated with high-dose melphalan followed by autologous stem cell transplantation in the era of novel agents, especially in the context of post-relapse survival and maintenance therapy with thalidomide, lenalidomide and bortezomib. Ninety consecutive transplant eligible patients at a single institution who were treated for multiple myeloma with highdose melphalan followed by autologous stem cell support were primarily included in the current study. Representative bone marrow mononuclear cell samples were available in 70 cases. Of the patients included in the current study, 29 received planned tandem ASCT. Patients received a mean of 1.4 lines of therapy prior to ASCT. All patients were subsequently analysed for RAS mutation status, which was correlated with clinical characteristics at diagnosis and therapeutic outcome. Table I summarizes the baseline and treatment characteristics for all patients included in the study.

Characterization of Human Leukemic HL-60 Sublines as a Model for Primary and Secondary Resistance Against Cytostatics

Resistance against cytostatics is a major problem in the therapy of hematologic malignancies. The intracellular alterations can be studied in vitro and several molecular mechanisms affecting all cellular levels like altered membrane transport rates (Pglycoprotein) [1], “trapping” of cytostatics in vesicles [2], detoxification (glutathione-S-transferase) [3], and alteration of nuclear target proteins (topoisomerases) [4] have been described. A special problem is the exhibition of a multidrug-resistant (MDR) phenotype, resulting in resistance against a broad spectrum of substances from different classes.

Different Functional States of Topoisomerase II can be Discriminated by Orthovanadate Sensitivity in Multi-drug Resistant Human Leukemic Cells

We have studied the functional properties of topoisomerase II (Topo II) in a subclone of the HL-60 cell line, which is highly resistant to cytotoxic Topo II inhibitors, but does not express p-glycoprotein. The cells contain the two forms of human topo II with Mr 170 and 180 kDa in equal proportions. Two different states of both forms of the enzymes can be separated by anion-exchange chromatography and functionally discriminated on the basis of orthovanadate sensitivity. The EC50 of orthovanadate was 0.2 microM for the early eluting and 30 microM for the late eluting Topo II.