First published online July 28, 2005
Stem Cells
Vol. 23 No.
9
October 2005, pp.
1417
-1422
doi:10.1634/stemcells.2005-0066; www.StemCells.com
© 2005 AlphaMed Press
Expression of Cytochrome P450 Genes in CD34+ Hematopoietic Stem and Progenitor Cells
Pavel Sou
eka,
Pavel Anzenbacherb,
Ivana Skoumalovác,
Michal Dvo
ákd
a Biotransformations Group, National Institute of Public Health, Center of Occupational Diseases, 
robárova, Czech Republic;
b Institute of Pharmacology, Faculty of Medicine, Palacky University at Olomouc and
c Hematooncological Department, Faculty Hospital Olomouc, Olomouc, Czech Republic;
d Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Key Words. CD34+ • Cytochrome P450 • Expression • Progenitor cells • Real-time polymerase chain reaction
Correspondence: Pavel Souek, Ph.D., Biotransformations Group, National Institute of Public Health, Center of Occupational Diseases, robárova 48, Praha 10, Czech Republic. Telephone: 420-267-082-711; Fax: 420-267-311-236; e-mail: psoucek{at}szu.cz
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ABSTRACT
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Expression of major cytochrome P450 forms (P450) was followed in preparation of purified hematopoietic CD34+ stem and progenitor cells. Levels of transcripts as well as mature proteins were traced by quantitative real-time polymerase chain reaction and by Northern and Western blotting. P450 1B1 and P450 2E1 proteins and respective mRNAs were found in all cases. On the other hand, no expression of P450 3A4, P450 3A7, and P450 2C9 was found. The results showed that expression of various P450 enzymes starts at different stages of cell differentiation. Both P450 forms found are known to be connected with cancer cells and with activation of procarcinogens (P450 1B1, polycyclic aromatic hydrocarbons; P450 2E1, nitrosamines, and solvents). Hence, cells at the early stage of differentiation already may be influenced by interaction with xenobiotics. This fact should also be taken into consideration when hematopoietic cell transplant therapy is applied.
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INTRODUCTION
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Cytochromes P450 (P450, EC 1.14.14.1
[EC]
) represent the most important group of biotransformation enzymes and play a key role in the metabolism of various endogenous chemicals (e.g., steroids), drugs of medical use, and environmentally related mutagens and carcinogens [1]. This study focused on the presence of four forms of P450 enzymes in CD34+ human hematopoietic stem and early progenitor cells (PBSPCs).
P450 1B1 activates many environmental mutagens and also catalyzes the 4-hydroxylation of estrogens, considered to be an important step in hormonal carcinogenesis [2]. It is expressed in several human tissues in which cancers typically occur such as prostate, ovary, uterus, and mammary gland [3].
P450 2E1 is known to be responsible for metabolic activation of many low-molecular-weight compounds, which are either toxic or suspected to act as chemical carcinogens (e.g., chlorinated hydrocarbons and nitrosamines [4]). P450 2E1 gene (CYP2E1) is expressed at the early stage of human fetus development, which indicates its importance for developing human organism [5]. It is one of the major hepatic P450 enzymes; however, it has also been detected at significant levels in human esophagus, kidney, lung [6], and brain [7].
P450 2C9 contributes not only to metabolism of drugs but also to biotransformation of fatty acids, prostanoids, and steroid hormones. It may catalyze potentially toxic bioactivation reactions [8]. P450 2C9 expression was found in the liver, kidney, adrenals, pancreatic islets, pituitary gland, lymphoid tissues, muscles, and epithelial cells in the skin, prostatic ducts, and gastrointestinal tract [9].
The steroid hydroxylase P450 3A4 is the most abundant P450 enzyme in the human liver, and P450 3A enzymes metabolize more than 50% of prescription drugs. The CYP3A4 is expressed in the liver, gut, colon, prostate, and breast [10]. Structurally similar, P450 3A7 is the major fetal form, which implies the role of P450 3A enzymes in early development [11].
The ratio of activation/detoxification of chemicals by P450s may modulate individual susceptibility to diseases such as cancer and influence therapeutic response to various drugs. Therefore, it is extremely important to assess expression and activity of these enzymes in biological systems for which use is considered in future human medicine. Stem cells are an excellent example among such systems [12]. The information about expression of genes coding for biotransformation enzymes, especially P450s in population of CD34+ human hematopoietic stem and progenitor cells, is still limited. To date, expression of mRNA for five cytochrome P450s (CYP1A1, 2A6/7, 2D6, 2E1, and 3A4) was demonstrated in human bone marrow, in various types of macrophages, in cell lines HL-60 and HEL, and in Epstein-Barr virus–transformed B-lymphoblastoid cell lines as well as in mononuclear cells [13, 14]. Presence of P450 2E1 in bone marrow cells was indicated by Western blotting [15]. The present study focused on bridging this gap in our knowledge. We have investigated gene expression and protein levels of four major P450 enzymes in a highly purified population of human CD34+ progenitor and stem cells (devoid of mononuclear leukocytes) obtained from peripheral blood.
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MATERIALS AND METHODS
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Materials
Chemicals for preparation of buffers were purchased from Sigma-Aldrich (Prague, Czech Republic, http://www.sigmaaldrich.com). Deoxynucleotides (dATP, dCTP, dGTP, and dTTP) for polymerase chain reaction (PCR) and molecular weight standards for electrophoresis (
X174DNA/HaeIII digest) were products of New England Biolabs, Inc. (Ipswich, MA, http://www.neb.com). UltraPure agarose was supplied by Life Technologies (Paisley, U.K., http://www.lifetech.com). Oligonucleotide primers were synthesized by Generi Biotech (Hradec Králové, Czech Republic, http://www.generi-biotech.com). Quantitative real-time PCR (QRTPCR) of CYP1B1 and CYP2E1 was performed using ThermoStart Polymerase Kit (ABgene, Surrey, U.K., http://www.abgene.com), and fluorescent probes were purchased from Integrated DNA Technologies (Coralville, IA, http://www.idtdna.com/Home/Home.aspx). Commercially available TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) including sets of specific probe and primers were used for QRTPCR of CYP2C9 (kit no. Hs 00426397_m1), CYP3A4 (Hs 00430021_m1), and cyclophilin A (4310857). TaqMan Universal PCR Master Mix (4304437; Applied Biosystems) was used for these assays as well.
Preparation of CD34+ Cells
Highly purified CD34+ stem and progenitor cells were prepared from peripheral blood of patients indicated for autologous transplantation of stem cells after mobilization with granulocyte colony-stimulating factor (G-CSF). The material used was an excessive material that was otherwise intended for disposal after patient death. Material from two patients was obtained. Both patients were asked to read and sign an informed consent before the treatment started. The procedure of autologous transplantation as well as the use of the material for research was approved by the Ethical Committee of Medical Faculty and of the Faculty Hospital, Palacky University, Olomouc. Separation method was based on two-step magnetic bead cell sorting using a Magnetically Assisted Chemical Separation (MACS) unit (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). Final preparation of PBSPCs was more than 96% of CD34+ cells and was devoid of mononuclear leukocytes and more differentiated cells of myeloid and lymphoid lineage as determined by flow cytometry by EPICS XL instrument (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com).
Isolation of Total RNA and cDNA Synthesis
Total RNA from CD34+ PBSPCs was isolated by Trizol according to the procedure supplied by the manufacturer (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). RNA quality and quantity was assessed by UV-VIS spectrophotometry on Carry 300 (Varian, Palo Alto, CA, http://www.varianinc.com/cgi-bin/nav?) and horizontal agarose gel electrophoresis. cDNA was then synthesized using 0.5 and 1 µg of total RNA with the help of First Strand cDNA Synthesis Kit purchased from MBI Fermentas (Vilnius, Lithuania, http://www.fermentas.com). Negative controls had the same composition as samples, but reverse transcriptase was omitted from the mixture. Quality of cDNA was then confirmed by PCR amplification of fragment from control gene ubiquitin C. Primers were designed to span first intron by help of MacOligo v4.0 program and GenBank database, forward (placed in exon 1): 5'-ATT TGG GTC GCA GTT CTT GT-3', reverse (placed in exon 2): 5'-TCC AGC AAA GAT CAG CCT CT-3'. Conditions for PCR amplification in GeneAmp 9700 thermocycler (Applied Biosystems) were as follows: 3 minutes of denaturation at 94°C, 35 cycles of 30 seconds at 94°C, 30 seconds at 64°C, 1 minute at 72°C, followed by final extension for 7 minutes at 72°C with 1.5 mM MgCl2 in the general reaction mixture. The resulting PCR product indicated whether cDNA derived from mRNA (190 bp) was contaminated by genomic DNA (1,009 bp).
QRTPCR Standards Preparation
As standards for absolute quantification of gene expressions, bacterial plasmids containing coding sequences of CYP1B1 (pCW'1B1-NPR), CYP2C9 (pCW'2C9-NPR), CYP2E1 (pCW'2E1), and CYP3A4 (pCW'NF14) were used. Plasmids were obtained as gifts from E.M.J. Gillam (Department of Physiology and Pharmacology, University of Queensland, Brisbane, Australia; pCW'1B1-NPR and pCW'2C9-NPR) and F.P. Guengerich (Center in Molecular Toxicology, Vanderbilt University, Nashville, TN; pCW'2E1 and pCW'NF14). Plasmid containing subcloned fragments of exons 1 and 2 of cyclophilin A in pCR2.1TOPO plasmid was kindly provided by D. Lison (Center of Occupational Medicine, Catholic University, Louvain, Brussels, Belgium). Plasmids were propagated in Escherichia coli DH5
Maximum Efficiency Cells (Invitrogen) and isolated by Plasmid Midi Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). After determination of concentration by spectrophotometry, plasmids were diluted by serial dilutions to 1,000,000, 100,000, 10,000, 1,000, and 100 copies per 5 µl and used in QRTPCR assay as calibration standards.
QRTPCR Assays
Method for determination of CYP2E1 gene expression was described elsewhere [16], and assay for CYP1B1 expression was developed as follows. Primers and probe were designed using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and MacOligo v4.0 programs. Sequences were then screened for mispriming by NCBI (National Center for Biotechnology Information) Blast (http://www.ncbi.nlm.nih.gov/BLAST) and for hairpins and loops by Mfold (http://mfold.burnet.edu.au). The resulting primer (forward: 5'-CCA GGA CAC CCT GTC CAC-3'; reverse: 5'-CCA CGA CCT GAT CCA ATT CT-3') and probe (5'-CAG GTA TCC TGA TGT GCA GAC T-3', FAM fluorophore at 5' end and BHQ1 quencher at 3' end) sequences were used for optimization of QRT-PCR assay. After varying cycling profiles, concentration of MgCl2, and concentrations of primers and probe, the following conditions were used in subsequent experiments. Final concentration of QRT-PCR components in 25 µl total volume was 5 mM MgCl2 (25 mM stock as part of the kit; ABGene), 0.2 mM dNTP mixture, 600 nM primers, 150 nM probe, 1.25 U ThermoStart Polymerase (5 U/µl stock as part of the kit; ABGene), and PCR buffer (10 times concentrated stock as part of the kit; ABGene). Twenty microliters of the above described mix was supplemented with 5 µl of Milli-Q ultrapure water (Biocel A10; Millipore, Billerica, MA, http://www.millipore.com) in negative controls, 5 µl of each standard stock solution in standards for calibration, or 5 µl of 10-times diluted cDNA solution after cDNA synthesis either in the presence or absence of reverse transcriptase (positive or negative samples). QRTPCR was performed in Rotor- Gene 2000 (Corbett Research, Sydney, Australia, http://www.corbettresearch.com) using the following cycling conditions: 15 minutes at 95°C to activate polymerase by hot start, 50 cycles of 10 seconds at 95°C, 10 seconds at 60°C, and 10 seconds at 72°C. Gain was set to 7, and fluorescence was acquired after elongation step. For TaqMan assays of CYP2C9 and CYP3A4, conditions described in producers protocols and brochures were used. Briefly, 10-times diluted cDNA samples or respective standards were amplified in TaqMan Universal PCR Master Mix. Milli-Q ultrapure water was added to adjust the total volume to 25 µl. The cycling conditions were as follows: 2 minutes at 50°C, 10 minutes at 95°C, 50 cycles of 15 seconds at 95°C, and 60 seconds at 60°C. Gain was set to 8, and fluorescence was acquired after elongation step. Results were evaluated by the program RotorGene v6 supplied by the producer of QRTPCR system (Corbett Research).
Immunoblotting
For Western blotting, the cells were thawed (if frozen), washed by isotonic phosphate-buffered saline (0.7% wt/wt sodium chloride in 0.1 M K/phosphate, pH 7.4), sonicated on ice for 10 seconds three times, and finally lysed by sodium dodecyl sulfate containing sample buffer for polyacrylamide electrophoresis (62.5 mM Tris-HCl, pH 6.8; 10% [vol/vol] glycerol, 4% [vol/vol] mercaptoethanol, 1% [wt/vol] bromophenol blue with 2% [wt/vol] sodium dodecyl sulfate). Protein concentration was determined in cell lysate by bicinchoninic acid method (Pierce, Rockford, IL, http://www.piercenet.com) After electrophoresis with a MiniProtean II apparatus (BioRad, Hercules, CA, http://www.bio-rad.com), the proteins were transferred by a Mini TransBlot electrophoretic cell of the same producer. For immunostaining, rabbit anti-human P450 1B1 (Alpha Diagnostics, San Antonio, http://www.4adi.com), P450 2E1, P450 3A4/7, and P450 2C9 (BD Gentest, Woburn, MA, http://www.gentest.com) antibodies were used. Chemiluminescence of the respective conjugates with alkaline phosphatase was followed with an ImmunStar (BioRad) detection kit.
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RESULTS
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Isolation of CD34+ PBSPCs, Total RNA, and cDNA Quality Assessment
Samples of CD34+ cells were prepared by magnetic bead sorting as described above. The final preparation contained 2.7 x 108 cells with purity over 96% as assessed by flow cytometry with fluorescein isothiocyanate–labeled antibodies. The total RNA was isolated from PBSPCs by Trizol procedure. The yield was 0.82 µg/µl. In total, 108 µg of total RNA was obtained with A260/280 ratio of 1.98 according to spectrophotometry. Agarose electrophoresis showed that RNA was pure and not degraded (no high-molecular-weight bands, distinct bands of 18S and 28S rRNA, no smears under 18S). Total RNA in amount of 0.5 and 1 µg was used for cDNA synthesis. Purity of cDNA was checked by PCR amplification of ubiquitin C fragment. This test proved that synthesized cDNA was present in full length and was not significantly contaminated by genomic DNA, as judged by absence of band of 1,009 bp in negative controls (Fig. 1
).
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Figure 1. Quality control of CD34+ cDNA using polymerase chain reaction amplification of ubiquitin C fragment. Lane 1, cDNA-negative control from 1 µg of total RNA; lane 2, cDNA-negative control from 0.5 µg of total RNA; lane 3, cDNA from 1 µg of total RNA; lane 4, cDNA from 0.5 µg of total RNA; lane 5, MW, X174DNA/HaeIII digest.
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QRTPCR Determination of Cyclophilin A and CYP Expression
Samples of PBSPC cDNA described above were diluted 10 times with Milli-Q ultrapure water and analyzed by QRTPCR. Assay of cyclophilin A expression was used as internal control of gene expression in various clinical samples [17, 18]. In the cells, cyclophilin A expression was very high, suggesting that during handling and processing of samples, no significant RNA degradation occurred (Table 1). Thus, expression levels of CYPs were determined in subsequent experiments. Results revealed very high expression of CYP1B1 (more than 750,000 copies/µg of RNA), which was in stem cells not reported so far (Table 1). CYP2E1 expression was moderate (less than 10,000 copies/µg of RNA), and expression levels of CYP2C9 and CYP3A4 were below the limit of quantification (Table 1). Reaction efficiency was consistent in all assays, and calibration curves showed very good linearity (Table 1). Also, performance of all tests in samples representing 0.5 and 1 µg of total RNA was satisfactory, including zero signals in negative cDNA controls. Control experiment showed no expression of CYP1B1 in breast cancer cell line MDA-MB-435, high expression in resistant breast cancer cell line NCI/ADR-RES, and moderate expression in intestinal Caco-2 cell line (results not shown). The expression of CYP2C9 and 2E1 varied from low to high in these cells (up to 20 times difference, results not shown). CYP3A4 expression in the three cell lines used was negligible.
Results of Immunoblotting Experiments
Western blotting was performed with cells washed and lysed as stated under Materials and Methods to liberate the P450 enzymes from membranes of endoplasmic reticulum. The procedure was sufficiently reliable to get samples yielding positive traces for P450 1B1 (Fig. 2A) in all samples studied as well as for the P450 2E1 (Fig. 2B). Under the same conditions, no expression of P450 2E1 was observed in human keratinocytes derived from HaCaT cell line serving as negative control (results not shown). On the other hand, no bands for P450 3A4/7 were observed. Also, no traces indicating the presence of the P450 2C9 protein were found, which is in line with the preliminary results [19] showing absence of the specific activity of P450 2C9 in the PBSPC microsomes. Hence, the Western blotting confirmed the expression of two P450 forms, P450 1B1 and P450 2E1, in the CD34+ PBSPCs. In parallel, the Northern blotting experiments were performed also, confirming the same results obtained with Western blotting as well as with the QRTPCR (presence of the CYP2E1 and CYP1B1 mRNA and absence of the CYP3A4, CYP3A7, and CYP2C9 mRNA in the sample of the PBPSC total RNA, results not shown).
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Figure 2. Immunoblotting of CD34+ cell lysate stained with antibodies against P450 1B1 (A) and P450 2E1 (B). (A): From left to right: lane 1, P450 1B1 standard (positive control); lanes 2–4, human liver microsomes (negative controls); lanes 5, 6, CD34+ cell lysate (two different protein concentrations, corresponding to 3 and 7 µg of protein/lane). (B): From left to right: lane 1, P450 2E1 standard (positive control); lane 2, human liver microsomes (positive control); lanes 3, 4, empty lanes; lanes 5–7, CD34+ lysate (5 µg of protein/lane in triplicate).
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DISCUSSION
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The data presented here are the first unequivocal results on P450 expression in human hematopoietic stem and progenitor cells at the early stages of differentiation. In both samples analyzed, the results of immunoblotting were the same. The patients were treated with G-CSF, which should not change the levels of the P450 enzymes [20]. As only two samples were available, any conclusions on the presence or absence of any interindividual variability in P450 content would be premature. The presence of P450 2E1 in bone marrow has been implicated by Bernauer et al. [15]. Hodges et al. [13] found five P450s, including P450 1A1, 2A6/7, 2D6, 2E1, and 3A4 in bone marrow, in macrophages, and in hematopoietic cell lines. Nguyen et al. [14] found even more P450 forms (16) in mononuclear fraction of peripheral blood. Clearly, in these papers, less-defined mixtures of cells ranging from pluripotent stem cells to already more-differentiated cells of hematopoiesis were involved. In this work, we demonstrate that in the earlier stages, only limited numbers of P450 forms are expressed. The presence of P450 1B1 and P450 2E1 is not surprising when their position in evolution trees of P450 enzymes is considered; also, their structures are rather conserved (http://drnelson.utmem.edu/CytochromeP450.html). Surprisingly, no P450 3A protein or mRNA was detected (in line with the absence of their specific activity, nifedipine oxidation [19]). This is particularly interesting for the P450 3A7 form, which is known to be present in human embryos and is downregulated during ontogenesis [11]. In the more differentiated cells of the hematopoietic origin, P450 3A form is apparently present, as has been shown in previously published studies [14, 21, 22]. Thus, our finding shows that the P450 3A enzymes are expressed lately during the differentiation of CD34+ stem cell population. The same probably also holds for P450 2C forms found by Nguyen et al. [14] and Hukkanen et al. [21].
Expression of P450 1B1 and 2E1 in human hematopoietic stem and progenitor cells in the early phase of hematopoietic cascade also has an implication for the safety of stem cell therapy in the future. In fact, both these forms are known to activate effectively known human precarcinogens to their ultimate forms (P450 1B1, polycyclic aromatic hydrocarbons; P450 2E1, nitrosamines, and organic solvents) [1, 2, 4]. Thus, exposure of both cells and patients to substrates metabolically activated by these enzymes should be avoided during cell mobilization with G-CSF, cell handling and storage, and post-transplantation recovery of patients.
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CONCLUSION
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Our results show expression of two environmentally important P450s but not of other pharmacologically active ones (P450 3A4/7, P450 2C9) in CD34+ hematopoietic stem and progenitor cells. The presence of some P450 forms in the earliest phase of hematopoiesis may also suggest that besides the known protective role of P450 enzymes, both expressed forms may have so far unknown essential roles in development and maturation of hematopoietic cells.
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ACKNOWLEDGMENTS
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We thank J. Baranova for help with RNA isolation. We also thank F.P. Guengerich (Center in Molecular Toxicology, Vanderbilt University) and E.M.J. Gillam (Department of Physiology and Pharmacology, University of Queensland) for their generous gifts (plasmids with CYP1B1, 2C9, 2E1, and 3A4 coding sequences). Plasmid-bearing fragment of human cyclophilin A was kindly provided by D. Lison (Center of Occupational Medicine, Catholic University, Louvain). The work at this project was financially supported by a grant from the Internal Grant Agency of Czech Ministry of Health (IGA NL 7295-3/2002).
DISCLOSURES
The authors indicate no potential conflicts of interest.
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Received on February 17, 2005;
accepted for publication on May 5, 2005.
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Abstract
Introduction
