Supplementary Data For

Supplementary Information

Letter to the Editor

Low doses of 2-deoxy-glucose sensitize acute lymphoblastic leukemia cells to

glucocorticoid-induced apoptosis

Karin Eberhart1,6*, Kathrin Renner1*#, Ireen Ritter1, Michael Kastenberger2, Katrin Singer3,

Claus Hellerbrand4, Marina Kreutz2, Reinhard Kofler5,6, Peter J. Oefner1

1Institute of Functional Genomics, University of Regensburg, Regensburg, Germany

2Department of Hematology and Medical Oncology, University of Regensburg, Regensburg, Germany

3Department of Internal Medicine 5 - Hematology/Oncology, University of Erlangen-Nuremberg, Erlangen,

Germany

4Department of Internal Medicine I, University of Regensburg, Regensburg, Germany

5Division of Molecular Pathophysiology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria

6Tyrolean Cancer Research Institute, Innsbruck, Austria

*These two authors contributed equally.

# Correspondence:

Dr. Kathrin Renner

Institute of Functional Genomics, Josef-Engert-Str. 9, 93053 Regensburg, Germany

T: +49-941-943-5008; F: +49-941-943-5019 E-mail: [email protected]

Table of Contents

Supplementary Figures S1 to S4

Supplementary Tables S1 to S4

Materials and Methods

References

1 Material and Methods

Cell culture, lymphocyte isolation and stimulation

The GC-sensitive cell lines CCRF-CEM-C7H2 T-ALL 1 and the 697/EU-3 cell line were used as model systems. The original name of the cell line 697/EU-3 given by its generator was

"line 697" 2 but it was recently renamed EU-3 by the original author 3. "697" is the term used for this cell line in repositories (like ATTC or DSMZ) where it is referred to as "human B cell precursor leukemia". It possesses t(1;19)/ translocation. The effect of 2-DG on p53 induced apoptosis was tested in the CEM-C7H2 subclone 4G5 that contains a temperature-inducible p53 expression vector 4. As GC-resistance models, CEM-C7H2-R9C10, CEM-C7H2-R1C57,

697/EU-3 R3C11, and R3D11 were used 5. Cells were maintained in RPMI 1640 supplemented with 10 % heat-inactivated tetracycline-free fetal calf serum, 2 mM L- glutamine (all PAA, Pasching, Austria) 100 units/ml potassium penicillin and 100 µg/ml streptomycin sulfate (Lonza, Verviers, Belgium) in a humified atmosphere of 95 % air, 5 %

CO2 at 37°C. All cell lines were regularly tested for mycoplasma contamination (Minerva

Biolabs, Berlin, Germany).

Primary lymphocytes were isolated by sequential leukapheresis, density gradient

(Ficoll/Hypaque), and countercurrent centrifugation (J6M-E centrifuge; Beckman, Munich,

Germany) 6. Polyclonally stimulated CD8+ T cell expansion cultures in RPMI 1640 medium supplemented with 10% human AB serum (PAN Biotech, Aidenbach, Germany) and 1.3 %

(v/v) T-Cell Growth Factor 7 (TCGF) were performed in 96-well U-bottom plates by adding

CD3/CD28 T-cell expander beads (Invitrogen, Karlsruhe, Germany) at 1 bead per cell to 8 x

104 CD8+ T cells once a week over 4 weeks. Reagents were obtained from Sigma (St. Louis,

MO, USA) unless indicated otherwise.

Induction and determination of apoptosis

Cells were diluted to 3 x 105 cells/mL and incubated with 2-DG (Calbiochem, San Diego, CA,

USA), gemcitabine, DEX, vincristine and anti-FAS antibody CH11 (Upstate Ltd, Dundee,

2 UK) at the indicated doses. Control cells were treated with carrier only, i.e., 0.1 % (v/v) ethanol in a 0.9 % (w/v) aqueous solution of NaCl. Expression of p53 in the CEM-C7H2 subclone 4G5 was induced at 32°C. For long-term experiments, reagents and medium were replenished every 48 h and cells diluted to 3 x 105 cells/mL. Apoptosis was determined by flow cytometric analysis of (i) the sub G1 fraction after DNA staining according to Nicoletti 8, and (ii) Annexin V/propidium iodide positive cells (Beckman-Coulter, Krefeld, Germany).

Cell number and protein determination

Cell number and volume were analyzed by means of a CASY1 TT cell counter (Schärfe

System, Reutlingen, Germany). Protein content was measured in the presence of a protease inhibitor (Pierce, Rockford, IL, USA) with the Coomassie Plus Protein Assay Reagent Kit

(Pierce) using bovine serum albumin as standard.

Cell fractionation

Six x 107 cells were harvested, washed with cold PBS, resuspended in 1 mL mitochondria isolation buffer (10 mM HEPES, pH 7.4, 0.25 M sucrose, 1 mM EDTA), and centrifuged at

500xg for 2 min at 4 °C. The supernatant was discarded and the pellet was resuspended in 1 mL isolation buffer. Cells were disrupted by the addition of digitonin at a concentration of 16 nmol/106. For CEM-C7H2, the mitochondria-enriched fraction was well separated from membranes, nuclei and cytosol by centrifugation at 13600xg for 12 min at 4 °C.

Functional analysis of mitochondria

Cellular oxygen consumption was measured with the SensorDish® Reader (PreSens Precision

Sensing GmbH, Regensburg, Germany). The optical oxygen sensors (OxoDish®) were luminescent dyes embedded in an oxygen-sensitive polymer at the bottom of each cell culture well. Signals were converted to % oxygen dissolved using the Presens software.

At indicated time points, respiratory chain function was analyzed by high-resolution respirometry (Oxygraph-2k, Oroboros, Innsbruck, Austria) at 37°C applying standard protocols. Cells were measured in a mitochondrial medium R05 9. Endogenous respiration

3 was measured after a stabilization time of 15 min, ATP synthase was inhibited by oligomycin

(2 µg/ml), and the capacity of the respiratory system was determined by uncoupling, applying p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP, 2 µM). Antimycin A inhibition was determined to test for non-mitochondrial respiration.

Measurement of enzyme activities

Activities of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured photometrically at 412 nm and 340 nm, respectively, using established protocols 9.

Metabolite quantification

Glucose and lactate (R-Biopharm, Darmstadt, Germany) concentrations in supernatants were measured enzymatically. Intracellular accumulation of 2-deoxy-glucose-6-phosphate was determined by CE-MS as described 10. Intracellular ATP was measured in viable cells by a luminescence assay (Promega, Madison, WI, USA) according to the manufacturer´s protocol.

Measurement of reactive oxygen species (ROS)

ROS were determined by 5-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-

H2DCFDA; Molecular Probes, Eugene, OR, USA) staining and FACS analysis.

Immunoblotting

Proteins were size-fractionated by SDS-PAGE and incubated with anti-hexokinase 1 (HK1,

Abnova, Taipei, Taiwan), anti-HK2 (Signaling Technology, Danvers, MA, USA), anti-COX-

IV (Novus Biologicals, Littleton, CO, USA), and anti-β-actin antibody. Secondary antibodies were horseradish peroxidase conjugates (GE Healthcare, Buckinghamshire, UK) and were visualised by an enhanced chemiluminescence reagent (Amersham, Uppsala, Sweden) on standard X-ray films. Films were scanned with a transmitted light scanner and band volumes were calculated with the Progenesis Same Spot software (Nonlinear Dynamics, Newcastle upon Tyne, UK). HK2 bands were normalized against β-actin or the mitochondrial loading control COX-IV.

Isolation of total RNA

4 Cells were lysed with 350 µL of Buffer RLT (Qiagen, Hilden, Germany) and homogenized using QIAshredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini

Kit (Qiagen). RNA quantity and purity were determined by optical density measurements

(OD260/280).

Real-Time RT-PCR

Reverse transcription was performed as described previously 11. First strand cDNA was synthesized from 1 µg of total RNA using Superscript Plus (Invitrogen). Expression of HK2

(MIM 601125) was analyzed applying the QuantiTect Primer Assay per the manufacturer's instructions (Qiagen) and normalized to β-actin (MIM 102630). Expression of GLUT1 (MIM

138140), GLUT3 (MIM138170) and GLUT14 (MIM611039) were analyzed on a

Mastercycler® ep realplex (Eppendorf, Hamburg, Germany) using the QuantiFast SYBR

Green PCR Kit (Qiagen) and normalized to 18S RNA.

Measurement of reactive oxygen species (ROS)

5 Two x 10 cells were washed with PBS twice, 300 µl of 0.2 µM CM-H2DCFDA were added, incubated for 20 min, the dye was removed and cells were analyzed. As positive controls, we used cells treated for 2 h with either rotenone (5 µM) or antimycine A (10 µM). FACS settings were calibrated with native and stained control cells.

Statistical analysis

For statistics the Mann-Whitney U test, the Wilcoxon test and the Student’s T-test were used.

Calculation of synergism

Synergism was determined by (i) calculating the Berenbaum factor 12 or (ii) summing up apoptotic rates induced by the single substances and comparing them to the apoptotic rates under the combined treatment in GC sensitive cell lines. For calculation of the Berenbaum factor, equipotent drug concentrations were determined and applied to the equation:

[DEXcombination treatment]/[DEXalone] + [2-DGcombination treatment]/[2-DGalone]. Values < 1 are considered as synergism, values = 1 indicate an additive effect and values > 1 an antagonistic effect.

5 Reference List

1 Strasser-Wozak EM, Hattmannstorfer R, Hala M, Hartmann BL, Fiegl M, Geley S et al.

Splice site mutation in the glucocorticoid receptor gene causes resistance to

glucocorticoid-induced apoptosis in a human acute leukemic cell line. Cancer Res.

1995; 55: 348-353.

2 Findley HW, Jr., Cooper MD, Kim TH, Alvarado C, Ragab AH. Two new acute

lymphoblastic leukemia cell lines with early B-cell phenotypes. Blood 1982; 60: 1305-

3 Zhou M, Gu L, Li F, Zhu Y, Woods WG, Findley HW. DNA damage induces a novel

p53-survivin signaling pathway regulating cell cycle and apoptosis in acute

lymphoblastic leukemia cells. J.Pharmacol.Exp.Ther. 2002; 303: 124-131.

4 Geley S, Hartmann BL, Hattmannstorfer R, Loffler M, Ausserlechner MJ, Bernhard D

et al. p53-induced apoptosis in the human T-ALL cell line CCRF-CEM. Oncogene

1997; 15: 2429-2437.

5 Schmidt S, Irving JA, Minto L, Matheson E, Nicholson L, Ploner A et al.

Glucocorticoid resistance in two key models of acute lymphoblastic leukemia occurs at

the level of the glucocorticoid receptor. FASEB J. 2006; 20: 2600-2602.

6 Andreesen R, Scheibenbogen C, Brugger W, Krause S, Meerpohl HG, Leser HG et al.

Adoptive transfer of tumor cytotoxic macrophages generated in vitro from circulating

blood monocytes: a new approach to cancer immunotherapy. Cancer Res. 1990; 50:

7450-7456.

6 7 Mackensen A, Carcelain G, Viel S, Raynal MC, Michalaki H, Triebel F et al. Direct

evidence to support the immunosurveillance concept in a human regressive melanoma.

J.Clin.Invest 1994; 93: 1397-1402.

8 Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple

method for measuring thymocyte apoptosis by propidium iodide staining and flow

cytometry. J.Immunol.Methods 1991; 139: 271-279.

9 Stadlmann S, Renner K, Pollheimer J, Moser PL, Zeimet AG, Offner FA et al.

Preserved coupling of oxidative phosphorylation but decreased mitochondrial

respiratory capacity in IL-1beta-treated human peritoneal mesothelial cells. Cell

Biochem.Biophys. 2006; 44: 179-186.

10 Timischl B, Dettmer K, Kaspar H, Thieme M, Oefner PJ. Development of a

quantitative, validated Capillary electrophoresis-time of flight-mass spectrometry

method with integrated high-confidence analyte identification for metabolomics.

Electrophoresis 2008; 29: 2203-2214.

11 Hellerbrand C, Muhlbauer M, Wallner S, Schuierer M, Behrmann I, Bataille F et al.

Promoter-hypermethylation is causing functional relevant downregulation of

methylthioadenosine phosphorylase (MTAP) expression in hepatocellular carcinoma.

Carcinogenesis 2006; 27: 64-72.

12 Berenbaum MC. Synergy, additivism and antagonism in immunosuppression. A critical

review. Clin.Exp.Immunol. 1977; 28: 1-18.