Per-Erik Sandström

Classification, incidence and survival analyses of children with CNS tumours diagnosed in Sweden 1984-2005

Aim:  Primary tumours in the central nervous system (CNS) are the second most common malignancy in childhood after leukaemia. Sweden has a high incidence and a high‐survival rate in international comparative studies. This has raised the question about the type of tumours included in the Swedish Cancer registry. We therefore compared international data to the Swedish Childhood Cancer registry.

Cell culture and Drosophila model systems define three classes of anaplastic lymphoma kinase mutations in neuroblastoma

SUMMARY Neuroblastoma is a childhood extracranial solid tumour that is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly requires characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs). Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and R1464STOP), which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T). In order to understand the potential role of these ALK mutations in neuroblastoma progression, we have employed cell culture-based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position 1174 (F1174I) generates a gain-of-function receptor capable of activating intracellular targets such as ERK (extracellular signal regulated kinase) and STAT3 (signal transducer and activator of transcription 3) in a ligand-independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALKF1174 mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i) gain-of-function ligand-independent mutations such as ALKF1174l, (ii) kinase-dead ALK mutants, e.g. ALKI1250T (Schönherr et al., 2011a) and (iii) ALK mutations that are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (Xalkori/PF-02341066), albeit with differing levels of sensitivity.

Measurements of serum glucose using the luciferin/luciferase system and a liquid scintillation spectrometer

A single-step assay for serum glucose measurements is described. The assay is based on the phosphorylation of D-glucose by glucokinase and the measurement of ATP consumption by firefly luciferase. The luminescence is recorded in an ordinary liquid scintillation spectrometer. The use of stable reagents and a stable final signal (light emission) makes it possible to analyze a large number of samples in each assay run. The assay is of particular value when repeated serum glucose determinations are performed on samples from small laboratory animals.

Identification of potassium flux pathways and their role in the cytotoxicity of estramustine in human malignant glioma, prostatic carcinoma and pulmonary carcinoma cell lines

Clinically-used drugs such as furosemide, bumetanide and cardiac glycosides, are modulators of transmembrane fluxes of cations. Recently, it has been suggested that the regulation of intracellular cation concentrations could be a primary target for anti-neoplastic drugs, and that the cytotoxic activity may be altered by inhibitors of cation fluxes at the level of the plasma membrane. Therefore, we investigated the mechanisms by which cations are translocated across the plasma membrane of malignant glioma (U251 MG), prostatic carcinoma (PC3) and pulmonary carcinoma (P31) cell lines. The interactions between cation flux inhibitors and the cytotoxicity of estramustine were also evaluated. Ouabain, the classical inhibitor of Na+, K+ATPase, markedly reduced 86Rb (K+) influx in all three lines, indicating that this ion transport system is present in the cells. Furosemide and especially bumetanide inhibited the 86Rb influx, indicating the presence of the Na+, K+, Cl- co-transport system. The potassium channel blocker, tetraethylammonium, but not apamin reduced the influx of 86Rb showing that high conductance K+ channels are present, but that channels of low conductance probably do not exist in these cell lines. The Na+, K+, Cl- co-transport inhibitors furosemide and bumetanide significantly reduced cytotoxicity of estramustine in P31 cells, whereas no interaction between other K+ flux inhibitors and the anti-neoplastic drugs were detected in any of the cell lines investigated. Thus, the data show that Na+, K+, ATPase and NA+, K+, Cl- co-transport systems and K+ channels of high conductance are present in malignant glioma (U251 MG), prostatic carcinoma (PC3) and pulmonary carcinoma (P31) cell lines, and that inhibition of the Na+, K+, Cl- co-transport system in P31 is associated with reduced cytotoxicity of estramustine. The results justify further studies evaluating the role of these cation flux pathways in terms of targets for anti-neoplastic therapy.

Volume regulation in mouse pancreatic β-cells is mediated by a furosemide-sensitive mechanism

A possible role for loop diuretic-sensitive Cl-/cation cotransport in volume regulation in the pancreatic beta-cells was investigated by measuring 86Rb+ efflux from beta-cell-rich pancreatic islets as well as the size of isolated beta-cells under different osmotic conditions. Lowering the osmolarity to 262 mosM (83% of control) resulted in a rapid cell swelling which was followed by regulatory volume decrease (RVD). RVD was completely inhibited by furosemide (1 mM), an inhibitor of Cl-/cation co-transport. The hypotonic medium (262 mosM) induced a rapid and strong increase in 86Rb+ efflux from beta-cell-rich mouse pancreatic islets and the furosemide-sensitive portion of the efflux was significantly increased. A slightly less hypotonic medium (285 mosM, 90% of control) induced only cell swelling and no RVD. With this medium only a marginal increase in 86Rb+ efflux was observed. Increasing the osmolarity by adding 50 mM NaCl (final osmolarity: 417 mosM, 132% of control) induced a rapid cell shrinkage but no regulatory volume increase (RVI). When the osmolarity was increased from a slightly hypotonic medium (262 mosM) to an isotonic medium (317 mosM) an initial cell shrinkage was followed by RVI. This RVI was inhibited by 1 mM furosemide. The data suggest that RVD as well as RVI in the beta-cells are mediated by loop diuretic-sensitive cotransport of chloride and cations and that these cells show a threshold for hypotonic stimulation of RVD.

Furosemide-induced glucose intolerance in mice is associated with reduced insulin secretion

The effect of furosemide on carbohydrate metabolism was studied in ob/ob mice. Intraperitoneal injection of a single dose of furosemide (200 mg/kg body weight) into fasted mice resulted in acute hyperglycaemia and two days after such a single dose, the mice showed fasting hyperglycaemia and glucose intolerance. Pancreatic islets from mice that had been injected with furosemide (200 mg/kg body weight) two days prior to the in vitro experiments showed increased basal (3 mmol/1 D-glucose) and decreased glucose-stimulated (20 mmol/1) insulin release. Islets from furosemide- or saline-injected animals showed no difference in islet insulin content. The results show that furosemide has both acute and long-term effects on carbohydrate metabolism in ob/ob mice. It is suggested that this, at least in part, is due to an effect on the pancreatic beta-cells.

Bumetanide reduces insulin release by a direct effect on the pancreatic β-cells

Abstract The effect of the loop diuretic bumetanide on glucose-induced insulin release, 45Ca2+ uptake, 36Cl− fluxes and 86Rb+ (K+ analogue) efflux was tested in isolated β-cell-rich mouse pancreatic islets. Low concentrations of bumetanide (0.1–10 μM) reduced glucose-induced insulin release as well as 45Ca2+ uptake. High concentrations (0.5–1) mM) augmented glucose-induced insulin release and an intermediate concentration (100 μM) had no effect. Bumetanide (0.01–1 mM) reduced the islet accumulation of 36Cl−. The net efflux of 36Cl− in the presence of 20 mM D-glucose was reduced by a concentration (10 μM) that lowered glucose-induced insulin release. Bumetanide (10 μM) did not affect the rate coefficient for 36Cl− efflux, which suggests that chloride permeability is not affected. Bumetanide (10 μM) reduced 86Rb+ efflux from preloaded islets. The data show that bumetanide reduces insulin release by a direct effect on pancreatic β-cells and suggest that this may be due to reduced chloide accumulation by a Na+, K+, Cl− co-transport system. It is suggested that the reduced chloride level is responsible for the decrease in glucose-induced chloride efflux and insulin release.

Inhibition by hydrochlorothiazide of insulin release and calcium influx in mouse pancreatic β‐cells

1 The effect of hydrochlorothiazide on insulin release, 36Cl− fluxes and 45Ca2+ uptake was tested in β‐cell‐rich mouse pancreatic islets. 2 At high glucose concentrations (10 and 20 mmol l−1), low concentrations of hydrochlorothiazide (0.1–1.0 μmol l−1) reduced insulin release by 22–42%. At lower glucose concentrations (3–8.5 mmol l−1) insulin release was not affected by the drug. 3 Neither short‐term influx (3 min) nor net accumulation (60 min) of 36Cl− in the islets was affected by hydrochlorothiazide (0.1–500 μmol l−1). 4 Glucose‐stimulated 45Ca2+ uptake was significantly reduced by hydrochlorothiazide (1–10 μmol l−1). 5 The data suggest that the diabetogenic effect of hydrochlorothiazide, at least in part, can be mediated by direct inhibition of insulin release from the pancreatic β‐cells. The inhibition is not mediated by reduced chloride fluxes but may rather be caused by inhibition of calcium uptake.

Na+ participates in loop diuretic-sensitive Cl−-cation co-transport in the pancreatic β-cells

In order to investigate whether Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the beta-cells, we tested the interaction between the effects of Na+ deficiency, furosemide and D-glucose on 86Rb+ fluxes in beta-cell-rich mouse pancreatic islets. Removal of extracellular Na+ slightly reduced the ouabain-resistant 86Rb+ influx and the specific effect of 1 mM furosemide on this influx was significantly smaller in Na(+)-deficient medium. The capacity of 20 mM D-glucose to reduce the ouabain-resistant 86Rb+ influx was not changed by removal of extracellular Na+. The 86Rb+ efflux from preloaded islets was rapidly and reversibly reduced by Na+ deficiency. Furosemide (1 mM) reduced the 86Rb+ efflux and the effect of the combination of Na+ deficiency and 1 mM furosemide was not stronger than the effect of furosemide alone. 22Na+ efflux was reduced by both ouabain and furosemide and the effects appeared to be additive. The data suggest that Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells. This adds further support to the idea that beta-cells exhibit a Na+, K+, Cl- co-transport system. Since some of the furosemide effect on 86Rb+ efflux persisted in the Na(+)-deficient medium, it is likely that also loop diuretic-sensitive K+, Cl- co-transport exists in this cell type.

Lupus anticoagulants in two children—bleeding due to nonphospholipid-dependent antiprothrombin antibodies

We describe two children with significant bleeding: one with multiple ecchymoses and the other with scrotal bleeding. In both patients, the activated partial thromboplastin time (APTT) was prolonged, with positivity for lupus anticoagulants (LA). However, the Owren prothrombin time (PT), usually insensitive for LA, was also prolonged. The presence of LA is associated with diverse clinical manifestations, with most patients being asymptomatic while others present venous or arterial thrombosis. Bleeding in conjunction with LA is rare and it is unusual to see prolongation of the Owren PT assay due to LA. An extended laboratory investigation of one of the patient’s plasma revealed not only LA but also a specific nonphospholipid-dependent antiprothrombin antibody causing an acquired hypoprothrombinemia. Conclusion: It is likely that the low prothrombin activity and not the LA caused the bleeding. The bleeding signs and symptoms in both patients subsided when the PT was normalized, although the prolonged APTT and the LA remained.