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Pitfalls in Detection of Contaminating Neuroblastoma Cells by Tyrosine Hydroxylase RT-PCR Due to Catecholamine-Producing Hematopoietic Cells

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Pitfalls in Detection of Contaminating Neuroblastoma Cells by Tyrosine Hydroxylase RT-PCR Due to Catecholamine-Producing Hematopoietic Cells ANTICANCER RESEARCH 26: 2075-2080 (2006) Pitfalls in Detection of Contaminating Neuroblastoma Cells by Tyrosine Hydroxylase RT-PCR Due to Catecholamine-producing Hematopoietic Cells ZYRAFETE KUÇI, GABRIELE SEITZ, SELIM KUÇI, HERMANN KREYENBERG, MICHAEL SCHUMM, PETER LANG, DIETRICH NIETHAMMER, RUPERT HANDGRETINGER and GERNOT BRUCHELT Department of Hematology and Oncology, Children’s University Hospital, D-72076 Tübingen, Germany Abstract. Background: RT-PCR analysis of compounds of contamination of hematopoietic stem cells by a low number catecholamine metabolism (in particular tyrosine of neuroblastoma cells. hydroxylase, TH) is widely used for the detection of contaminating neuroblastoma cells in hematopoietic stem The contamination of hematopoietic (stem) cells by cell preparations. Due to reports in the literature showing neuroblastoma cells is often analyzed by tyrosine hydroxylase that hematopoietic cells are also able to produce (TH)-RT-PCR, presuming catecholamine metabolism to be a catecholamines, we investigated whether TH-RT-PCR is specific marker for neuroblastoma (1-9). However, two really suitable for this purpose. Materials and Methods: potential problems should be considered with this approach. Besides neuroblastoma cells, mononuclear blood cells, The expression of catecholamine markers in neuroblastoma is apheresis preparations and hematopoietic stem cells were heterogeneous. Some neuroblastoma cells are able to produce used for single and nested RT-PCR. In addition to TH, the dopamine and noradrenaline, some produce only dopamine expressions of dopamine-‚-hydroxylase and noradrenaline and some of them generate only the precursor, DOPA. transporter were analyzed. Results: Using single RT-PCR, a Furthermore, there is also a heterogeneous expression of the clear discrimination between neuroblastoma and hemato- uptake system for catecholamines, dopamine and poietic cells was possible. However, by using nested RT-PCR, noradrenaline transporters. Figure 1 gives a summary of the the "neuroblastoma markers" were also detected in a enzymes and uptake proteins that are, in principle, significant percentage of non-mobilized mononuclear blood characteristic of neuroblastoma cells. Since the expressions of cells, in mononuclear blood cells of healthy donors the mRNAs and the respective proteins for catecholamine mobilized with G-CSF, and in highly purified CD34+ and synthesis and uptake are variable at different stages of the cell CD133+ stem cells from healthy mobilized donors. cycle and are influenced by cytokines and other substances, it Conclusion: Our results raise the question of whether the is advantageous to use more than one catecholamine marker. RT-PCR analysis of compounds of catecholamine Therefore, in addition to TH, two other markers of metabolism is suitable and selective enough to detect the catecholamine metabolism were used in order to circumvent potential heterogeneous gene expression: dopamine-‚- hydroxylase (DBH) and the noradrenaline transporter (NAT), which is responsible for the uptake of [123I]mIBG, widely used Abbreviations: DBH, dopamine-‚-hydroxylase; DOPA, 3,4 dihydroxy- for scintigraphic imaging of neuroblastoma cells (10). phenylalanine; G-CSF, granulocyte-colony stimulating factor; MACS, However, an even more severe problem in the sensitive and magnetic-activated cell sorting; NAT, noradrenaline transporter; NB, neuroblastoma; PBMNC, peripheral blood mononuclear cells; specific detection of neuroblastoma cells by RT-PCR may be RT-PCR, reverse transcriptase polymerase chain reaction; TH, the fact that hematopoietic cells are also able to produce and tyrosine hydroxylase; PBSC, peripheral blood stem cells. take up catecholamines, as was recently reported by several groups (11-18). Correspondence to: Dr. Gernot Bruchelt, Department of Hematology The aim of this study was, therefore, to evaluate whether and Oncology, Children’s University Hospital, Hoppe-Seyler-Str.1, RT-PCR of TH (and other markers of catecholamine D-72076 Tübingen, Germany. Tel: (0049-7071) 2984710, Fax: (0049- metabolism) is suitable for the specific and sensitive 7071) 295482, e-mail: [email protected] determination of neuroblastoma cells in hematopoietic stem Key Words: Tyrosine-hydroxylase, RT-PCR, neuroblastoma, cell transplants. For this purpose, single and nested RT-PCR hematopoietic stem cells (CD34+/CD133+). analysis were used. 0250-7005/2006 $2.00+.40 2075 ANTICANCER RESEARCH 26: 2075-2080 (2006) (pH 8.5), 75 mM KCl, 3 mM MgCl2, 10 mM dithiotreitol, 125 ÌM dNTP(each) and 40 U RNaseOUTì]. Single (First) PCR. Two microliters of the first-strand cDNA were used as the template for the PCR reaction performed in 50 Ìl mixture, containing 10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 ÌM dATP, dGTP, dTTP and dCTP (Invitrogen), 20 pmol of amplimers (each sense and antisense) for GAPDH, TH, noradrenaline transporter (NAT) and DBH and 1.25 U AmpliTaq Gold Polymerase (Applied Biosystems, Kassel, Germany). The reaction conditions were: 5 min at 95ÆC, followed by 28 cycles (GAPDH) and 38 cycles ( TH, NAT and DBH): 96ÆC for 15 sec, 55ÆC for 30 sec and 72ÆC for 90 sec. At the end, the reaction mixture was kept for 7 min at 72ÆC and finally chilled at 4ÆC until analysis. Primer sequences for single PCR: GAPDH: sense 5’-GAC AAC AGC CTC AAG ATC ATC-3’, antisense 5’-GAC GGC AGG TCA GGT CCA CCA-3’, 325 bp; TH: sense 5’-TGT CAG AGC TGG ACA AGT-3’, antisense 5’-GAT ATT GTC TTC CCG GTA GC-3’, Figure 1. Catecholamine synthesis and re-uptake in neuroblastoma cells 299 bp; NAT: sense 5’-GCT TCT ACT ACA ACG TCA TCA TC-3’, (DOPA: 3,4 dihydroxyphenylalanine; DA: dopamine; NA: noradrenaline; antisense 5’-CGA TGA CGA CGA CCA TCA G-3’, 294 bp; DBH: TH: tyrosine hydroxylase; AADC: aromatic amino acid decarboxylase; sense 5’-ATC CAC ATC TTC GCC TCT CAGC-3’, antisense DBH: dopamine-‚-hydroxylase; DAT: dopamine transporter; NAT: 5’-TCT TCC GTG TTG TAC GTG CAG G-3’, 206 bp. noradrenaline transporter). Nested PCR. Two microliters of the first PCR product for GAPDH were amplified by 28 cycles, whereas the number of cell cycles for Materials and Methods TH, DBH and NAT was 38. The reaction conditions were the same as for the first PCR. Blood cells. Peripheral blood mononuclear cells (PBMNC) and G-CSF-mobilized PBMNC from healthy donors were prepared by Primers for nested PCR. GAPDH: sense 5’-AAT GCC TCC TGC Ficoll-separation. Peripheral blood stem cells (PBSC) (CD34+ and ACC ACC-3’, antisense 5’-ATG CCA GTG AGC TTC CCG-3’, 247 CD133+) were obtained from the mobilized peripheral blood of bp; TM: sense 5’-GTT CGA CCC TGA CCT GGA CT-3’, antisense healthy donors and were purified using magnetic-activated cell sorting 5’-TGT ACT GGA AGG CGA TCT CA-3’, 95 bp; NAT: sense 5’- (MACS) with a progenitor cell isolation kit (Miltenyi Biotech, TCT TCT CCT CCT TCA CCC TCA ACC-3’, antisense 5’-GAC Bergisch Gladbach, Germany ) as described (2, 19). ACC ACG CTC ATA AAA CTC GGC-3’, 167 bp; DBH: sense 5’- ACA CAC CTG ACT GGG AGA AAG G-3’, antisense 5’-CAC Human neuroblastoma cell lines. SK-N-SH [American Type Culture GAC CTT CTT CAA CAT GCG G-3’, 127 bp. Collection (ATCC, MD, USA), Kelly [German Collection of The amplicons were applied to a polyacrylamide gel (8% Microorganisms and Cell Cutures (DSMZ; Braunschweig, Germany)] acrylamide, 0.25% bis-acrylamide). After electrophoresis, the gels and SiMa cells were obtained from P. Marini [Children's University were stained with ethidium bromide and visualized with ultraviolet Hospital Tübingen, Germany (20)]; LS were established by R. illumination, digitally imaged by the CS-1 Videoimager (Cybertech, Handgretinger, [Children’s University Hospital Tübingen, (21)]; Berlin, Germany). SK-N-LO were obtained from Helson (22). Neuroblastoma cell lines were cultured in RPMI-1640 supplemented with 10% (v/v) fetal bovine Results serum (FBS), 2 mM glutamine, 100 U/ml penicillin and 100 Ìg/ml streptomycin, in a 5% CO2 / 95% humidified atmosphere at 37ÆC. Summary of RT-PCR expression of catecholamine markers in human neuroblastoma cell lines and neuroblastoma tissues. RNA samples of neuroblastoma patients. Samples (19 before and 18 Table I gives an overview of the RT-PCR expression of the after chemotherapy) were obtained from Dr. Beck, Children's University Hospital Tübingen. Neuroblastoma tissue was kindly three catecholamine markers (TH, DBH and NAT) used in provided by Dr. Schilling and Dr. Treuner (Olga Hospital, Stuttgart, all experiments of this study in five neuroblastoma cell lines. Germany). The table documents the results of the single RT-PCR (usually 28 and 38 cycles, respectively) obtained repeatedly RT-PCR analysis. Total RNA was extracted from neuroblastoma cell over a period of several years. Although the expressions of lines and hematopoietic cells by using the RNeasy mini kit (Qiagen, these genes varied, depending on the metabolic status of the Hilden, Germany) with DNase digestion using RNase-Free DNase cell lines (e.g., sometimes very low expression of TH in Set, according to the manufacturer’s instructions. cDNAs were reverse transcribed from 2 Ìg total RNA with 0.5 Ìg oligo(dT)12-18 SK-N-SH cells), the well-established human neuroblastoma Primer and 200 U SuperScriptTM II RNase H-Reverse Transcriptase cell lines SK-N-LO and LS strikingly never expressed the (Invitrogen, Karlsruhe, Germany) according to the supplier’s respective genes under the experimental conditions used in protocol. [Reaction volume: 20 Ìl, containing 50 mM Tris-HCl buffer this study. 2076 Kuçi et al: Pitfalls in Tyrosine Hydroxylase RT-PCR Analysis Table I. RT-PCR expression of TH, DBH and NAT in human Table II. RT-PCR expression of TH, DBH and NAT in neuroblastoma neuroblastoma cell lines. tissue from 37 patients. Cell lines TH NAT DBH Stage TH NAT DBH SK-N-SH + + + I (n=11) 8* 8* 8* Kelly + + + II (n=7) 7 7 7 SiMa + + + III (n=8) 8 8 8 SK-N-LO - - - IV (n=10) 10 10 10 LS - - - IVs (n=1) 1 1 1 n=5-10; single RT-PCR.
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