Stem Cells, Vol. 16, No. 2, 136-143,
March 1998
© 1998 AlphaMed Press
Identification by Differential Display of Transcripts Regulated during Hematopoietic Differentiation
H.M. Bonda,
P. Bonellia,
M. Mesuracad,
V. Agostib,
C. Masonea,
C. Cuomoa,
A. Nisticòd,
P. Tassoned,
F. Tuccilloa,
L. Ceccoa,
L. Iacopinoc,
V. Barbierid,
M. Cerraa,
F.S. Costanzod,
G. Morroned,e,
S. Venutad,e
a Department of Experimental Oncology, National Cancer Institute, Fondazione G. Pascale, Napoli, Italy;
b Department of Biochemistry and Medical Biotechnology, Federico II University, Napoli, Italy;
c Department of Hematology-Oncology, Azienda Ospedaliera "Bianchi-Melacrino-Morelli," Reggio Calabria, Italy;
d Department of Experimental and Clinical Medicine, University of Catanzaro, Italy;
e CEINGE Advanced Biotechnology, Napoli, Italy
Key Words. Hematopoietic progenitors • Leukemia • Differential display • Polymerase chain reaction • Gene regulation
Correspondence:
Prof. S. Venuta, Department of Experimental and Clinical Medicine, Faculty of Medicine, University of Catanzaro, Via T. Campanella Catanzaro, Italy, I-88100.
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Abstract
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The polymerase chain reaction-based differential display method (DDRT-PCR) was used to identify mRNAs differentially expressed during the maturation of human CD34+ progenitor cells stimulated to differentiate in vitro towards granulomonocytic or erythroid lineages with a mixture of hemopoietins (kit ligand + interleukin 3 + GM-CSF in the absence or presence of erythropoietin, respectively). Three cDNA transcripts (B32, B41, and B56) display differential expression during cytokine-induced maturation of CD34+ cells. These clones have no homology with already-described sequences. Primer extension confirmed the presence of the corresponding mRNA. The levels of mRNA corresponding to B32 are enhanced in the later phases of the granulomonocytic as well as in the erythroid differentiation of CD34+ cells. The mRNA identified by B41 was induced by a late stage in only granulomonocytic differentiation of CD34+ cells. The mRNA corresponding to B56 was instead present in nonstimulated CD34+ cells, declined in the early stages of differentiation, and reappeared at later stages in cells treated with both combinations of cytokines. Expression of these genes was detected in a number of acute myelogenous leukemias, as well as in some leukemic cell lines. B32 and B41 were downregulated in KG-1 cells induced to differentiate towards the monocytic lineage, whereas the levels of B56 were unchanged. In K562 cells, clones B41 and B56 were downregulated only in the late phases of PMA-induced megakaryocytic differentiation and during erythroid differentiation. B32 was rapidly downregulated when K562 cells were induced to differentiate towards either megakaryocytic or erythroid phenotypes. These transcripts represent novel hematopoietic cDNAs that should prove of value for the study of human blood cells and their disorders.
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Introduction
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The differentiation of hematopoietic stem and progenitor cells involves a series of molecular changes that result in the progressive loss of self-renewal ability and pluripotency, and in the parallel acquisition of specialized functions characteristic of mature blood cells. The molecular events that govern this process are still only partially understood; however, a number of genes specifically expressed in progenitor CD34+ cells have been identified and characterized and may be required for hematopoietic differentiation [1-5]. Inappropriate expression of such genes or modifications in the structure of their products may result in alterations of normal hematopoiesis and development of leukemia [6, 7].
So far, owing to limited availability of hematopoietic progenitors, most of the studies performed to identify hematopoietic regulatory molecules have exploited the existence of human cell lines, mostly of leukemic origin, which can be induced to differentiate in vitro by specific stimuli, in a manner that mimics to some extent "physiological" hematopoiesis [8]. However, the development of effective purification procedures and of polymerase chain reaction (PCR)-based methods has rendered the direct analysis of gene expression in hematopoietic progenitors feasible.
This study was aimed at identifying genes whose expression is regulated during hematopoietic differentiation. The technique of differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) [9] was used to compare the mRNA profiles from human progenitor cells at various stages of cytokine-induced differentiation. We report here the isolation of three novel cDNA clones whose expression appears to be regulated during the differentiation of human CD34+ cells in vitro. These transcripts are also present in a number of leukemic samples, and their levels are modulated during the differentiation of the leukemic cell lines KG-1 and K562.
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Materials and Methods
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CD34+ Cells
Hematopoietic progenitor cells (CD34+) were isolated from either human umbilical-cord blood or from peripheral blood of cancer patients treated with human recombinant hemopoietins by either immunopanning with MicroCELLector flasks (Applied Immuno Science; Menlo Park, CA) as described elsewhere [10], or using the miniMACS system QBEND/10, Miltenyi (Biotech GmbH; Milano, Italy). In both cases, the cells were 80%-95% positive for CD34 as assessed by flow cytometry, and the ability to differentiate was determined by in vitro clonogenic assays. Viable contaminating cells included mainly erythroblasts and T and B lymphocytes. CD34+ cells were cultured in Iscove's modified Dulbecco's medium (Flow; Milano, Italy) containing 20% fetal calf serum (FCS), c-kit ligand ([KL], 10 ng/ml, Sigma; Milano, Italy), interleukin 3 ([IL-3], 25 U/ml), and granulocyte-monocyte colony-stimulating factor ([GM-CSF], 50 U/ml) in the presence or absence of erythropoietin ([Epo], 2 U/ml), all from Boehringer-Mannheim (Milano, Italy), for different time points up to 15 days.
Cell Lines
All cell lines were cultured in RPMI 1640 medium (Flow) containing 10% FCS (Hyclone; Kent, UK), 100 U/ml of penicillin, 100 µg/ml streptomycin and 2 mM glutamine (Flow). The factor-dependent cell line GF-D8 cells was maintained in the presence of 30-50 ng/ml of recombinant GM-CSF. The differentiation-inducing reagents, PMA (phorbol 12-myristate 13-acetate, "TPA") ionomycin, sodium butyrate and bovine hemin were all from Sigma. For stimulation of differentiation, the KG-1 cells were treated with PMA 10–7M and ionomycin (1.6 µg/ml), whereas the K562 cells were maintained in the presence of either PMA 10–8M, sodium butyrate 1 mM, or hemin 5 x 10–5M.
Peripheral Blood Leukocytes
Mononuclear cells from peripheral blood of healthy volunteers (PBMNC) were isolated by Ficoll-Hypaque density gradient centrifugation and were cultured for three days in the presence of 1 µg/ml phytohemagglutinin-P (PHA) Sigma.
Leukemic Cells
Leukemic blasts from peripheral blood or bone marrow of patients affected by acute myelogenous leukemias were isolated by Ficoll-Hypaque (Pharmacia; Milano, Italy) density gradients. Normal leukocytes generally accounted for less than 5%-10% of the total population. Cells were analyzed by flow cytometry for phenotype determination, and aliquots were used for RNA extraction.
Preparation of RNA
RNA was prepared according to the method of Chomczynski and Sacchi [11]. When a small number of cells were used, lysis was carried out in a reduced volume of guanidinium thiocyanate buffer (200 µl) followed by phenol/chloroform extraction, including 10 µg of glycogen as a carrier. To remove chromosomal DNA, the RNA samples were digested with 0.5 U of DNase (RNase-free, Promega; Florence, Italy) in the presence of 1 U of RNase inhibitor (Promega) for 20 min at 37°C, followed by 65°C for 20 min. To verify the effective removal of chromosomal DNA, amplifications were carried out with specific primers on mock-reverse transcribed RNA samples. For some experiments, poly A+ RNA was isolated using oligo dT columns (Pharmacia).
Differential Display
DDRT-PCR was either performed essentially as described by Sokolov and Prockop [12], where two random defined decamers were used for amplification, or as described by Liang et al. [13], where one decamer and either dT12VG or dT12VC were used (V indicates a variable base either C, G, or A). cDNA was synthesized from aliquots of the CD34+ cell preparations at 0, 3, 6-7, and 12-15 days of differentiation with KL, IL-3, GM-CSF, and Epo. The primers used were either 5 pmol of random hexamers (Boehringer-Mannheim) or 20 pmol of dT12VG or dT12VC. After annealing was performed at 65°C for 20 min, cDNA was synthesized with Superscript II reverse transcriptase (GIBCO BRL; Milano, Italy) for 60 min at 37°C. This cDNA was quantified for the amount of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), by RT-PCR prior to DDRT-PCR. The amplifications were performed in duplicate and contained PCR buffer (10 mM Tris/HCl, pH 8.3, 50 mM KCl, 1.2 mM MgC12), 2 µM dNTP, 1-3 µCi 
32P dATP, either 2 decamers (50 pmol) or dT12VG or dT12VC (50 pmol) with one decamer (50 pmol) and 0.2 U Taq polymerase (Polymed; Florence, Italy) in a volume of 20 µl. The random defined decamers (50% CG/AT ratio) were as described in the differential display kit (Display Systems, Tandil Ltd; Paris, France). Samples were overlaid with mineral oil and the amplifications performed in a Gradient Robocycler (Stratagene; Heidelberg, Germany). The conditions for amplification after an initial denaturation at 95°C for 5 min, were: denaturation at 95°C for 30 sec, annealing at 36°C (random primed cDNA) or 40°C (dT12VG or dT12VC primed cDNA) for 1 min, and elongation at 72°C for 1 min 30 sec, for 40 cycles, followed by 5 min extension at 72°C.
Amplification products were displayed on 6% nondenaturing polyacrylamide gels [14]. Gels were dried and used to expose x-ray film (Kodak; NEN, Milano, Italy) for 24-48 h. The bands differentially expressed were excised, rehydrated in 200 µl H20 and boiled for 15 min. Two µl of each band were reamplified as above using 200 µM dNTP for 40 cycles, and the products were gel-purified and cloned into the pGEM-T vector (Promega). Clones containing inserts of the correct size were sequenced using the T7 sequencing kit (Pharmacia). Sequences were screened against nucleic acid data bases with FASTAsearch [15] and/or BLASTsearch [16].
RT-PCR with Specific Primers
Specific primers were prepared corresponding to clones which had no homology with known genes and which were at least 250 bases in size. The primers were designed to have 18-23 bases with a melting temperature (Tm) in the range 56°-60°C. For each couple of primers (forward and reverse), the amplified PCR product was checked for the expected size and purified from the gel to be used as a template for radiolabeling. cDNAs prepared from RNA samples were normalized using the levels of GAPDH as an internal control. Amplification conditions were set which resulted in product that could be detected by hybridization with specific probes but not by ethidium bromide staining (non-maximal), and were estimated to be in a linear range. Amplifications were performed using 10 pmol specific primers, 200 µM dNTP, 0.1U Taq in PCR buffers for 20-22 cycles for GAPDH and IL-2R-
c [10], 30 cycles for the CD34 mRNA, and 40 cycles for the clones tested. Amplifications were either performed singly or as coamplifications where, for example, B32 was first amplified for 20 cycles and then for a further 20 cycles with the addition of primers specific for GAPDH, Taq, and dNTP. The products were then analyzed by agarose gels in duplicate and hybridized separately with the different probes. Once amplified, the samples were electrophoresed on 1% agarose gels and after alkali denaturation transferred onto Nytran filters (Schleicher and Schuell; Dassel, Germany). Filters were incubated at 80°C under vacuum for two h, hybridized overnight at 65°C in Church buffer with 1-2 x 107 cpm of radiolabeled probe, extensively washed, and used to expose x-ray film for 24-48 h. The probes corresponding to each amplification were prepared by amplifying from a purified template (or from the plasmid pGEM-T containing the cloned DDRT-PCR bands) in the presence of 50 µM dGTP, dCTP, dTTP, and 10-30 µCi 32P dATP, and 5 pmol of primers for 15 cycles.
Northern Blotting
Probes labeled as for RT-PCR (2-4 x 107 cpm) from either specific amplified templates or from the plasmid were hybridized at 65°C in Church buffer to Northern blots containing RNA (15 µg of total RNA per lane) from a panel of cell lines: HPB-All, H9, CEM, MOLT4, Jurkat, DAUDI, HEL, MO7, U937, HL60, KG-1, K562, MC3, and HeLa.
Primer Extension
Primer extension reactions were performed by annealing 40 µg total RNA prepared from K562, KG-1 cells, or tRNA with either 10 pmol specific primer or 10 ng amplified denatured template in hybridization buffer (40 mM PIPES pH 6.4, 1 mM EDTA, 0.4 M NaCl, and 80% formamide) at 56°C for 18 h. After ethanol precipitation, a reverse transcriptase reaction was carried out with Superscript II in the presence of 5 µCi 32P dATP at 42°C for 60 min. The cDNA was treated with RNase, phenol extracted, ethanol precipitated, and analyzed on 6% polyacrylamide denaturing gels.
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Results
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Differential Display
DDRT-PCR was performed to comparatively analyze a selection of the mRNA repertoire of CD34+ cells stimulated with KL, IL-3, GM-CSF, and Epo for 0, 3, 6, and 12 days. An example of DDRT-PCR is shown in Figure 1. Two different preparations of CD34+ cells are used for DDRT-PCR, either from umbilical cord blood or from peripheral blood of patients treated with recombinant cytokines; these stem cell preparations display very similar profiles. In this experiment, several minor bands and one major band were apparently regulated during differentiation.
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Figure 1. DDRT-PCR performed using a CD34+ progenitor differentiation time course. The cDNA samples used for DDRT-PCR were obtained from CD34+ cells prepared from umbilical cord blood (CB) or peripheral blood (PB) from cancer patients treated with recombinant cytokines at T = 0. CD34+ cells from peripheral blood were stimulated with KL, IL-3, GM-CSF, and Epo for 0, 3, 6, and 12 days. Amplifications were performed with two decamers at 36°C for annealing, and the products are displayed on a 6% polyacrylamide gel and used to expose x-ray film for 24 h.
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Using a total of 40 different combinations of primers, 83 bands were selected which appeared to be differentially expressed. These bands have been reamplified, cloned, and sequenced. Ten of these represented repetitive elements, eight were mitochondrial sequences, and several were homologous to already-described genes. These genes included some which are known to be found in hematopoietic cells—for example, the chain of the IL-3 receptor [2-4]; topoisomerase IIß, which catalyzes the interconversion of topological isomers of DNA and is expressed in hematopoietic cell lines and leukemias [17] and is indeed a target of antineoplastic reagents; the adducin chain [18], which has been described to be present in the erythrocyte cytoskeleton [19]; and annexin II, which is a molecule widely expressed and described to have a role in the secretory process of neutrophils [20]. In addition, 16 sequences corresponded to expressed sequence tags (EST), sequences determined from the 5' or 3' ends of randomly chosen cDNA library clones within the framework of the Human Genome Project [21]. The majority of clones obtained showed no homology to those contained in nucleic acid data banks.
Characterization of the Cloned Bands
The expression of cDNAs was studied in CD34+ cells induced to differentiate, hematopoietic cell lines, and leukemias by RT-PCR using specific amplimers (Table 1). In particular, we concentrated on characterizing the expression of three bands which appear to be regulated during hemopoietin-induced differentiation of CD34+ cells. B32 (309 bp) and B41 (273 bp) were derived from DDRT-PCR performed with two decamers, and B56 (304 bp) was from DDRT-PCR performed with T12VG and one decamer (Table 1). Figure 2 illustrates the specific amplification (RT-PCR) of these three cloned bands on CD34+ cells induced to differentiate by using different cytokine cocktails, either towards myeloid (KL, IL-3, GM-CSF) or erythroid (KL, IL-3, GM-CSF, and Epo) phenotypes. The amounts of cDNA are normalized for GAPDH; as expected, the amount of mRNA for the CD34 antigen in the progenitor cells decreases dramatically during differentiation. Amplification specific for B32 appears toward the end of both time courses (myeloid and erythroid); the transcript corresponding to B41 instead appears only in the myeloid differentiation (days 6-13), suggesting a lineage specificity. B56 is present in the CD34+ cells before stimulation; its level then drops by days 1 and 3 to reappear to a higher level by day 6 and day 13, in response to both the myeloid and erythroid stimulation in these time courses.
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Figure 2. RT-PCR analysis of differential display derived clones in time courses of CD34+ cells stimulated to differentiate. Purified CD34+ cells (peripheral blood) were stimulated either in the myeloid (M) (IL-3, KL, and GM-CSF) or in the erythroid (E) direction (IL-3, KL, GM-CSF, and Epo) for time points up to 15 days. From RNA preparations, RT-PCR was performed using primers for GAPDH (22 cycles) as a control for amounts, primers for CD34 (30 cycles), and specific primers for the bands (B32, B41, and B56, 40 cycles). Amplified products were detected by hybridization with the respective radiolabeled probes.
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These clones were tested for expression by RT-PCR in a variety of leukemia-derived hematopoietic cell lines (Table 2). All three mRNAs were present in the cell lines KG-1 and K562, which both display relatively immature features; B41 and B56 were also expressed in some B- and T-lymphoid cell lines, whereas the expression of B32 was restricted only to cell lines with myeloid phenotype (KG-1, K562, HEL, DAMI, and MO7).
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Table 2. RT-PCR analysis of the expression of differential display obtained clones in different hematopoietic cell lines
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The KG-1 cell line can be induced to differentiate in vitro towards the monocytic lineage by treatment with PMA in combination with the Ca++ ionophore, ionomycin, resulting (Fig. 3A) in a dramatic decrease in the expression of the mRNA for the progenitor-specific antigen, CD34, as seen in Figure 2, and as has been documented [1]. B32 and B41 were both downregulated following differentiation of KG-1, whereas the amount of B56 was unchanged. IL-2R c was used to monitor for PMA-induced differentiation in K562, as has been documented [10] where the mRNA and protein expression are strongly upregulated (Fig. 3A). Erythroid differentiation induced by butyrate or hemin was evident as the amount of ferritin H mRNA was upregulated by Northern blot analysis (not shown). When K562 cells were stimulated to differentiate towards megakaryoblastic (PMA) or erythroid phenotypes (butyrate or hemin), B32 was in both cases rapidly downregulated. B41 and B56 were only decreased at later time points of PMA treatment, whereas they were both downregulated in a similar fashion to B32 in response to butyrate or hemin. These trends were confirmed when the clones were coamplified with GAPDH in KG-1 and K562 time courses (Fig. 3B).
These mRNAs were also tested for expression in a panel of acute myelogenous leukemias (Table 3). B56 was present in all cases, showing a higher level of expression in the more immature M1 type, whereas B32 and B41 were present in 12 and 11 of the 17 cases of AML, respectively. For B32 and B41 the distribution of expression relative to the FAB classification was less evident but they were particularly present in M1 cases. No apparent association emerged between the expression profile of these genes and that of the CD34 antigen. Normal PBMNC as well as activated PBMNC had low (B56) or no detectable expression.
None of the three cDNAs gave any signal when tested by Northern blot with a panel of hematopoietic cell lines. However, the amount of material available from the CD34+ cells or leukemias was not sufficient to permit Northern analysis. Total RNA (35 µg) and poly A+ mRNA (5 µg) from KG-1 and K562 cells were tested by Northern blotting with probes specific for B32, B41, and B56, but no significant hybridization was detected after 10 days of exposure (not shown), indicating low abundance of the mRNA transcripts. This must reflect a low expression of these transcripts consistent with the results of the RT-PCR analysis, where 40 cycles of amplification and then hybridization with a radiolabeled probe are required to detect amplification of these RNAs. Primer extension experiments were therefore performed to confirm the presence of corresponding mRNAs. Figure 4 shows the extended products from cDNA prepared from K562, primed with either the entire cDNA fragment or specific primers; the products were extended for up to approximately 700 bp for B41 and 600 bp for B56, indicating a primer-specific elongation. A similar primer extension profile was obtained for B56 from KG-1 and GF-D8 cells (results not shown). For B32, primer extension gave a weak but distinct band at approximately 660 bp (results not shown).
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Figure 4. Primer extension analysis of B41 and B56. Primer extension reactions were performed by annealing 40 µ g of K562 RNA or tRNA with either the forward primer for B41 or the entire cDNA fragment, forward or reverse primers (as in Table 2) for B56. Reverse transcription was performed in the presence of 32P dATP and the denaturing gels autoradiographed for 24 h. Size markers indicated are radiolabeled pBR322 digested with HinfI. The radiolabeled amplified fragments for B41 and B56 are also shown.
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The expression patterns observed by RT-PCR indicate that the B32, B41, and B56 transcripts are predominantly expressed towards the early to intermediate stages of CD34+ cellular differentiation, but before the cells attain a fully mature phenotype. This is consistent with the finding that in KG-1 and K562 cells, which may be regarded as a counterpart of progenitor-precursor cells at specific stages of maturation, these genes are downregulated in response to stimuli that induce differentiation.
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Discussion
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The DDRT-PCR technique offers two main advantages for the study of hematopoietic stem cells: first, it permits analysis of very small amounts of mRNA, thereby enabling the study of fresh progenitors instead of hematopoietic cell lines; second, it allows a comparative analysis of variations in subsets of the mRNA repertoire of hematopoietic progenitors undergoing the different stages of differentiation. The DDRT-PCR method has been applied to hematopoietic cells with some success; for example, CD34+/CD38+ progenitor cells have been compared with the more immature CD34+/CD38– cells [22], resulting in the identification of a novel cDNA sequence specific to the CD34+/CD38– population. Similarly, a comparative analysis of myelodysplastic patients with normal controls using the DDRT-PCR technique yielded a cDNA fragment shown to be downregulated in the patients [23].
The three cDNA transcripts obtained from progenitor stem cells are characterized as being regulated in the hematopoietic system, they all appear to correspond to low-abundant mRNAs, and the presence of mRNA for these was verified by primer extension. Expression of these bands is found in the early or intermediate stages of differentiation of CD34+ cells towards the granulomonocytic and/or erythroid lineages. B41 appears to be lineage-specific, induced only in the granulomonocytic pathway, whereas B32 is expressed during both granulomonocytic and erythroid differentiation. B56 is instead expressed also in unstimulated CD34+ cells but repressed during the early stages of differentiation. The levels of these transcripts are also regulated during the differentiation of KG-1 and/or K562 cells, respectively, and in all cases they decline with the induction of maturation. Since these cell lines display features of relatively immature progenitor cells, and as, unlike the fresh progenitors in culture, they represent a homogeneous population and can be induced to progress through discrete steps of maturation by treatment with appropriate stimuli, these cells can be regarded as an advantageous parallel system to investigate the regulation of the expression of the transcripts studied.
Another interesting finding is the presence of significant levels of all three RNAs in samples of AML cells with a relatively immature phenotype, (M1), compared to PBMNC and AMLs with more mature features. This hints at a possible involvement of these genes in mechanisms of leukemogenesis and further supports the idea that a significant regulated expression of these cDNA transcripts occurs in specific stages of hematopoiesis.
Based on the above results, it would be tempting to speculate that the expression of these mRNAs may be required at specific stages of maturation of hematopoietic progenitor/precursor cells and then decline when the cells become fully differentiated. Indeed, the levels of mRNAs for a variety of hematopoietic regulatory molecules are known to be subjected to stringent transcriptional and/or post-transcriptional control. However, additional information on the molecules encoded by these transcripts will be necessary to assess their role in normal and malignant hematopoiesis. None of these clones displayed a significant degree of homology with any of the sequences contained in DNA data bases, thereby suggesting that they correspond to novel mRNAs.
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Acknowledgments
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The authors wish to acknowledge the technical assistance of Mrs. Rita Bisogni, Mrs. Rita Sorice, Mr. Luigi Spiezia, Mr. Alfredo De Rosa and Mr. Domenico Esposito. Supported in part by funds from A.I.R.C. (Associazione Italiana per la Ricerca sul Cancro), C.N.R. (Consiglio Nazionale delle Richerche), F.S.N. (Fondo Sanitario Nazionale), and M.U.R.S.T. (40% and 60%).
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accepted for publication on January 21, 1998.
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Abstract
Introduction
