ANTICANCER RESEARCH 26: 2075-2080 (2006)

Pitfalls in Detection of Contaminating Neuroblastoma Cells by Hydroxylase RT-PCR Due to -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 that hematopoietic cells are also able to produce (TH)-RT-PCR, presuming catecholamine metabolism to be a , 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 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.

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(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. n=number of neuroblastoma tissues. TH=tyrosine hydroxylase; NAT=noradrenaline transporter; Numbers in columns TH, DBH, NAT: positive samples. DBH=dopamine-‚-hydroxylase. *Very low expression in 3 out of 11 cases. TH=tyrosine hydroxylase; NAT=noradrenaline transporter; DBH=dopamine-‚-hydroxylase.

The RT-PCR expression of the respective genes of Table III. Expression profiles of hematopoietic cells from healthy donors neuroblastoma tissue from patients with different (nested RT-PCR). neuroblastoma stages are shown in Table II. In all samples TH NAT DBH investigated, the three markers could be detected in tissues of stage II, III, IV and IVs patients. However, in three out of PBMNC (n=9) 5/9 2/2 4/5 eleven patients suffering from neuroblastoma stage I the Aphersis prep. (n=3) 1/3 2/3 1/3 markers could not be clearly identified. Generally, there was a Non-adherent cells (n=4) 0/4 3/4 2/4 large variation in the expression levels of these markers in the Stem cells (n=10) CD34+ (n=3) 2/3 2/2 1/2 different samples. CD133+ (n=7) 2/7 4/4 0/4

Comparison of RT-PCR expression of TH, DBH and NAT in n=number of samples. neuroblastoma cells and PBMNCs. In order to directly Quotients present the number of positive preparations / total number of analyzed preparations. compare the RT-PCR expressions of TH, DBH and NAT in TH=tyrosine hydroxylase; NAT=noradrenaline transporter; hematopoietic and neuroblastoma cells, PBMNCs from DBH=dopamine-‚-hydroxylase; PBMNC=peripheral blood mononuclear healthy donors and the neuroblastoma cell line Kelly were cells. used. The Kelly cells proved to constantly express the markers at high levels. In single RT-PCR analysis, TH, DBH and NAT could not be detected in PBMNCs. The method allows the detection of about one neuroblastoma cell in 100-1000 PBMNCs from four healthy non-mobilized persons. DBH hematopoietic cells (data not shown). In order to enhance the signals were detected in three of them; TH was positive in all sensitivity of detection, nested RT-PCR was used. Often, four cases presented in this figure. In two of them (lanes 7 and conflicting results were obtained (i.e., expression of the 9), additional signals were detected. It is known that at least catecholamine markers in PBMNC from healthy donors). seven splice variants of TH exist (23), and the additional signals However, this is in line with recent reports which in lanes 7 and 9 might represent such variants. In any case, TH demonstrated that hematopoietic cells were also able to signals could be clearly detected by nested RT-PCR in more produce and take up catecholamines (11-18). Therefore, we than 50% of the nine PBMNC samples investigated (Table III). analyzed this problem systematically and confirmed that In further experiments, the expressions of the catecholamine hematopoietic cells of a significant number of non-mobilized markers in hematopoietic stem cells from ten mobilized healthy donors expressed catecholamine genes. Figure 2 shows healthy donors were analyzed (Table III). In Figure 3b, the the RT-PCR pattern in first (a) and nested (b) RT-PCR of RT-PCR expression of TH, DBH and NAT is shown in a PBMNCs compared to Kelly cells. In the first RT-PCR, sample of CD34+ hematopoietic stem cells after first and catecholamine signals were never observed in hematopoietic nested RT-PCR. In first RT-PCR, TH, DBH and NAT were cells (up to 38 cycles). However, nested PCR showed the negative (lanes 2-4). However, all three signals were positive presence of these signals in a significant number of PBMNC under nested RT-PCR conditions (lanes 5-7). samples from healthy donors (see also Table III). Summary of the expression profiles of hematopoietic cells from Nested RT-PCR of different hematopoietic cell preparations. healthy donors by nested RT-PCR. In Table III, the expression Figure 3a shows the nested RT-PCR pattern (TH, DBH) of pattern obtained from PBMNCs, apharesis preparations and

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Figure 2. a: Single RT-PCR of neuroblastoma (Kelly cells) and PBMNC. Kelly cells (28 cycles): lane 1: MW marker; lane 2: GAPDH; lane 3: NAT; lane 4: TH; lane 5: DBH; lane 6: empty. PBMN (38 cycles): lane 7: GAPDH; lane 8: NAT; lane 9: TH; lane 10: DBH. b: Nested RT-PCR of neuroblastoma (Kelly cells) and PBMNC. Kelly cells (28 cycles): lane 1: MW marker; lane 2: GAPDH; lane 3: NAT; lane 4: TH; lane 5: DBH; lane 6: empty; PBMNC (38 cycles); lane 7: GAPDH ; lane 8: NAT; lane 9: TH; lane 10: DBH.

Figure 3. a: Nested RT-PCR (TH, DBH) of PBMNC from four healthy non-mobilized donors (first PCR: 28 cycles; nested RT-PCT: 38 cycles). Lane 1: MW markers; lane 2: DBH person A; lane 3: DBH person B (negative); lane 4: DBH person C; lane 5: DBH person D; lane 6: TH person A; lane 7: TH person B; lane 8: TH person C; lane 9: TH person D. b: Nested-RT-PCR (TH, DBH, NAT) of CD34+ hematopoietic stem cells from a mobilized healthy donor (first PCR: 28 cycles; nested RT-PCR: 38 cycles; lanes 2-4: first PCR; lanes 5-7: nested PCR). Lane 1: MW markers; lane 2: GAPDH (positive) and DBH (negative); lane 3: TH (negative); lane 4: NAT (negative); lane 5: GAPDH (upper) and DBH (lower); lane 6: TH; lane 7: NAT.

hematopoietic stem cells (CD34+ and CD133+) are samples. However, neuroblastoma cells have a high affinity to summarized (n=26). Taken together, we found positive metastasize into the bone marrow and, therefore, purging signals for TH in 38%, NAT in 87% and DBH in 44% of procedures were developed in order to remove contaminating blood cell samples tested. neuroblastoma cells, e.g., by ex vivo treatment with 6-hydroxydopamine (6-OHDA), but this approach was not Discussion successful (24). Progress was made with the development of procedures for the isolation of hematopoietic stem cells by One approach to cure metastasizing neuroblastoma is magnetic separation with iron-loaded antibodies, which allows autologous transplantation of hematopoietic stem cells. In the the isolation of hematopoietic stem cells with high purity (19). past, the first attempts were made using bone marrow However, none of these procedures can absolutely exclude

2078 Kuçi et al: Pitfalls in Tyrosine Hydroxylase RT-PCR Analysis contaminating neuroblastoma cells in the stem cell catecholamine metabolism is not an unequivocal parameter in preparation. Therefore, it is useful to develop methods which order to precisely distinguish hematopoietic stem cells from allow a very sensitive determination of few neuroblastoma or neuroblastoma cells, even if a broader panel of catecholamine other cancer cells among a huge excess of hematopoietic cells. markers is used. Nevertheless, it should be considered that The amplification of cancer-specific gene expression using several other research groups have argued that RT-PCR (e.g., RT-PCR is most suitable for this purpose in principle. as real-time RT-PCR) is suitable for the detection of However, very often it is a difficult issue to find a suitable gene neuroblastoma cells using TH or other markers of which is exclusively expressed in cancer cells and not in catecholamine metabolism (1-9). However, in actual cases, we hematopoietic cells. The hallmark of neuroblastoma as a tumor were not able to differentiate with certainty whether a positive of the sympathetic peripheral nervous system is its ability to signal stemmed from contaminating neuroblastoma cells or produce, metabolize and take up catecholamines. Therefore, from the hematopoietic cells themselves, using the test especially the rate-limiting enzyme of catecholamine synthesis, conditions described. The only way to get comparable results in TH, which catalyses the transformation of tyrosine to different laboratories is to stringently standardize the protocols 3,4 dihydroxyphenylalanine (DOPA), has been widely used for concerning the analysis procedures, storage, isolation of cells the identification of these cells (1-9). However, TH is not and RNA, chemicals and mRNA markers (27, 28). Due to the necessarily expressed in all neuroblastoma cells, and some of fact that catecholamine metabolism also plays an important them preferentially use tyrosinase instead of TH for the role in hematopoietic cells, it appears that catecholamine hydroxylation of tyrosine to DOPA (25). On the other hand, markers are not ideal candidates even if the amounts of expression of the TH gene can vary considerably, depending on expressed mRNAs in neuroblastoma and hematopoietic stem the cell cycle, and is influenced by other parameters (cytokines, cells are markedly different. Finally, whether the knowledge of retinoic acid etc). In order to avoid these problems and enhance a slight neuroblastoma cell contamination, e.g., after purging, the diagnostic safety, two other parameters of catecholamine has any consequences for the treatment and survival of patients metabolism, DBH and the NAT were included. The occurrence should be discussed. Of course, it is a convincing idea that an of at least one of these three mRNAs should be a strong hint of autologous transplantation without contaminating the presence of neuroblastoma cells among an excess of neuroblastoma cells is superior, and many reports argue that hematopoietic cells. Indeed, using single RT-PCR conditions this is indeed the case. However, at least in one publication for the successful detection of neuroblastoma cells, no signals (29), the fact that slight contamination of transplanted could be detected in hematopoietic cells (see Figure 2a). hematopoietic stem cells with neuroblastoma cells may even However, when neuroblastoma cells were mixed with be advantageous for survival, due to priming and activation of hematopoietic cells (PBMNC), one neuroblastoma cell could immune competent cells which could destroy neuroblastoma be detected among 100-1000 hematopoietic cells using the test cells that survived the former treatment is mentioned. system described. Apparently, this sensitivity is not enough to detect even slight contamination with neuroblastoma cells. Acknowledgements Therefore, nested RT-PCR was used, further enhancing the sensitivity to about 100 to 1000-fold. Surprisingly, we often Supported by fortüne-Project 1012, University of Tübingen, and observed clear signals for TH, DBH or NAT in hematopoietic Deutsche Krebshilfe, grant 10-2082-Br1, Germany. cells from healthy persons who certainly were not contaminated with neuroblastoma cells (Figure 3 and Table III). TH signals References were also detected by another group in a small number of blood samples (6%) not contaminated with neuroblastoma cells, but 1 Pession A, Libri V, Sartini R, Conforti R, Margini E, Bernardi they were interpreted as being false-positive (26). In our L, Fronza R, Olivetto E, Prete A, Tonelli R and Paolucci G: Real-time RT-PCR of tyrosine hydroxylase to detect bone opinion, the occurrence of these signals is based on the fact marrow involvement in advanced neuroblastoma. Oncol Rep 10: that hematopoietic cells can, to a certain extent, also produce, 357-362, 2003. metabolize and take up catecholamines (11-18). Consequently, 2 Lode HN, Handgretinger R, Schuermann U, Seitz G, Klingebiel T, they also have to express genes for the corresponding enzymes Niethammer D and Beck J: Detection of neuroblastoma cells in or transport proteins, even if they are present at much lower CD34+ stem cells using a combination of tyrosine hydroxylase levels than in "professional" catecholamine-producing cells. RT-PCR and anti-ganglioside GD2 immunohistochemistry. Eur J Signals for TH, DBH and NAT were not observed in Cancer 33: 2024-2030, 1997. hematopoietic cells by using first RT-PCR (i.e., under poorly- 3 Tsang KS, Li CK, Tsoi WC, Leung Y, Shing MM, Chik KW, Lee V, Ng MH and Yuen PM: Detection of micrometastases of sensitive conditions), but these signals were observed in many neuroblasrtoma to bone marrow and tumor dissemination to cases when using nested RT-PCR (i.e., under sensitive hematopoietic autografts using flow cytometry and reverse conditions). According to our results and the data from the transcriptase-polymerase chain reaction. Cancer 97: 2887-2897, literature, it is concluded that RT-PCR using mRNAs of 2003.

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