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Derangement of transcription factor profiles during in vitro differentiation of HL60 and NB4 cells
Leukemia Research 31 (2007) 827–837
Derangement of transcription factor profiles during in vitro differentiation of HL60 and NB4 cells Malene Bjerregaard Pass, Niels Borregaard, Jack Bernard Cowland ∗ The Granulocyte Research Laboratory, Department of Hematology 93.2.2, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Received 23 June 2006; received in revised form 23 June 2006; accepted 16 July 2006 Available online 30 August 2006
Abstract Sequential up- and down-regulation of a handful of critical transcription factors is required for proper neutrophil differentiation. Malfunction of transcription factors may lead to diseases such as acute myeloid leukemia (AML) and specific granule deficiency. In order to understand the molecular background for normal and malignant granulopoiesis, a good model system is required that faithfully mimics the in vivo transcription factor expression profiles. The two human leukemic cell lines HL60 and NB4 have been widely used as model cell lines for these purposes. Differentiation of HL60 and NB4 cells resulted in asynchronous differentiation to morphologically mature neutrophils over a period of 5–7 days. To obtain cell populations of more even maturity, cells at different stages of in vitro differentiation were purified by immunomagnetic isolation. This resulted in three cell populations that could be classified as promyelocytes, myelocytes/metamyelocytes, and mature neutrophils, respectively. Comparison of transcription factor mRNA profiles from these cell populations with those previously seen in normal human bone marrow, demonstrated that although all of the 14 transcription factors described in vivo, could be detected during in vitro differentiation, vast differences in their expression profiles was observed. These data illustrate the limitations of cell lines as models for normal granulopoiesis. © 2006 Elsevier Ltd. All rights reserved. Keywords: HL60; NB4; AML; Granulopoiesis; Transcription factors; Granule proteins; C/EBP; PU.1
1. Introduction Formation of the neutrophil granulocyte takes place in the bone marrow. A number of transcription factors play a critical role during this process and mutations of transcription factor genes may lead to aberrant maturation of neutrophils. Chromosomal translocations involving Runx1 (also called AML1) [1], and mutations or deregulation of C/EBP␣ [2–4] is frequently observed in acute myeloid leukemia (AML) patients. Furthermore, mutations of PU.1 have in some cases been associated with AML [5] and disruption of C/EBP has been observed in patients with specific granule deficiency [6]. Gene disruption studies in mice have also identified several transcription factors essential for neutrophil devel∗
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0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2006.07.019
opment. Lack of definitive hematopoiesis is observed in Runx1−/− or c-myb−/− fetuses, demonstrating their importance for all hematopoietic lineages, including neutrophils [7–9]. Mice deficient in C/EBP␣ completely lack mature neutrophils and eosinophils, whereas all other hematopoietic lineages are present, and function normally [10]. Immature myeloid precursor cells are found in the blood of C/EBP␣ knock-out mice, demonstrating that C/EBP␣ is essential for neutrophil differentiation [10]. Conditional knock-outs of C/EBP␣ have demonstrated that C/EBP␣ is essential for the transition from the common myeloid progenitor (CMP) to the granulocyte/macrophage restricted progenitor (GMP), but not for GMPs to terminally differentiate to mature neutrophils [11]. C/EBP-deficient mice also fail to develop fully mature neutrophils and eosinophils, only atypical hyposegmented neutrophils were present [12]. Transcripts for azurophil granule proteins were detected in the bone marrow, whereas an
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almost complete lack of transcripts for specific granule proteins was noted [12–14]. Mice deficient in PU.1 also generate neutrophils with a block in differentiation [15–17]. Conditional knock-outs have demonstrated that PU.1 is essential for the generation of CMPs, and for the terminal maturation of GMPs to mature neutrophils [18,19]. Analysis of knock-out mice is valuable if one wish to demonstrate the requirement of a given transcription factor for myeloid differentiation. However, due to the inherent property of knock-out studies this method only allows one to determine the earliest point of differentiation where the transcription factor in question is essential. Furthermore, as it is becoming evident that not only the presence of a transcription factor, but also its overall concentration [20,21] – as well as its concentration relative to that of other factor transcription factors [22] – is important for lineage decision and differentiation, model systems to investigate these issues are also required. Cellular systems to study transcription factor regulation during myeloid differentiation in vitro may therefore be of great value provided the models faithfully mimick the in vivo profile of transcription factor expression. Two human leukemic cell lines, HL60 and NB4, are widely used as models for neutrophil differentiation. The HL60 cell line was established in 1977 from a patient suffering from AML, FAB M2 [23]. The cells grow continuously as myeloblasts and can differentiate to morphologically mature looking neutrophils after addition of all-trans retinoic acid (ATRA) and dimethyl sulfoxide (DMSO) [24]. The differentiation is not complete since specific granules and gelatinase granules are not formed and transcripts for matrix proteins contained in these granules are not detectable [25,26]. Membrane proteins of these granules are synthesized but are routed to the plasma membrane [27]. Constitutive expression of the specific granule protein NGAL in HL60 cells results in an accumulation of NGAL in the azurophil granules when the cells are at the myeloblast stage. However, when differentiation of HL60 cells is induced, newly synthesized NGAL is routed extracellularly and does not accumulate in granules, reflecting the inability of more mature HL60 cells to synthesize granules and retain granule proteins [25,27]. The NB4 cell line was established in 1991 from a patient suffering from acute promyelocyte leukemia (APL) having the t(15;17) translocation [28]. Morphologically, the cells are characterized as promyelocytes and can be stimulated to differentiate to neutrophils by ATRA. Like HL60 cells, specific and gelatinase granules are not formed during in vitro differentiation of NB4 cells and mRNA for specific granule matrix proteins cannot be detected [29]. Also like in HL60 cells, membrane proteins of specific and gelatinase granules are synthesized when neutrophil maturation is induced and routed to the plasma membrane [30]. We have previously used a method for separation of neutrophil precursors from human bone marrow into three populations of different maturity. These are myeloblasts (MB) + promyelocytes (PM), myelocytes (MC) + metamyelocytes (MM), and band cells (BC) + seg-
mented neutrophil cells (SC), respectively. By analyzing these populations by Northern and Western blotting, a highly individualized expression of 14 transcription factors important for neutrophil differentiation was demonstrated during in vivo granulopoiesis [31]. The purpose of this study was to correlate the expressionpattern of transcription factor mRNAs in HL60 and NB4 cells during in vitro differentiation, to the in vivo pattern referred to above and to determine whether these two leukemic cell lines may be used as model systems for transcription factor regulation during granulopoiesis.
2. Materials and methods 2.1. In vitro differentiation of HL60 and NB4 cells HL60 cells (CCL-240) were obtained from American Type Culture Collection (ATCC) and NB4 cells were generously provided by Dr. M. Lanotte [28]. The cells were cultured in RPMI 1640 with glutamax-1 (Invitrogen, San Diego, CA, USA) supplemented with 10% fetal calf serum (FCS) (Invitrogen), 100 units/ml penicillin (Invitrogen), and 100 g/ml streptomycin (Invitrogen). In vitro differentiation of HL60 was induced by addition of 10−6 M ATRA (Sigma–Aldrich, St. Louis, MO, USA) and 1.3% DMSO and of NB4 by addition of 10−6 M ATRA. 2.2. Purification of cells by immunomagnetic isolation Exponentially growing NB4 or HL60 cells were depleted for spontaneously differentiated cells expressing CD11b by addition of anti CD11b antibody (BD Pharmingen, San Diego, CA, USA) followed by MACS beads, anti-IgG1 (10 l/107 cells) (Miltenyi Biotech, Bergisch Gladbach, Germany), and depleted using an AS column (Miltenyi Biotech). We refer to these depleted populations as “day 0 cells”. At day 2 (NB4) or day 3 (HL60) of the in vitro differentiation, cells expressing CD11b were positively selected. The cells were incubated with anti CD11b antibody followed by MACS beads, anti-IgG1 (10 l/107 cells), and positively selected on a BS column (Miletenyi Biotech). We refer to these positive selected populations as “day 2 cells” and “day 3 cells”. At day 6 of in vitro differentiation, cells still expressing CD49d were depleted. The cells were incubated with anti CD49d antibody (BD Pharmingen) followed by MACS beads, antiIgG1 (10 l/107 cells), and depleted on a BS column. We refer to these depleted populations as “day 6 cells”. In vitro differentiation and immunomagnetic isolation was performed twice for both cell lines with very similar results—the result of one experiment is shown. 2.3. Flow-cytometric analysis For flow-cytometric analysis the cells were stained with anti CD11b-PE (mouse IgG1 , BD Pharmingen), CD49d-PE
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(mouse IgG1 , BD Pharmingen), or isotype control. Cells were incubated with antibody for 15 min at room temperature for labeling, washed twice in 0.5% BSA in PBS, and fixed in 1% paraformaldehyde in PBS. The cells were analyzed on a FACScan instrument (Becton Dickinson, San Jose, CA, USA). Data was analyzed using the CellQuest software. 2.4. Northern blotting Total RNA was isolated with Trizol (Invitrogen). The RNA was ethanol-precipitated and resuspended in 0.1 mM EDTA. For Northern blotting, 5 g of RNA was run on a 1% agarose-gel and transferred to a Hybond-N membrane (GE Healthcare, Chalfont ST. Giles, United Kingdom) as described [31]. Filters were pre-hybridized for 1–2 h at 42 ◦ C in 6 ml ULTRAhyb (Ambion, Austin, TX, USA) and hybridized overnight at 42 ◦ C after addition of further 4 ml containing the 32 P-labeled probe and sheared salmon sperm DNA (10 g/ml). The membranes were washed as described [31] and developed by a Fuji BAS2500 PhosphorImager. Membranes were stripped by boiling in 0.1% SDS before re-hybridization. The probes are described previously [31]. They were radiolabeled with [␣-32 P] dCTP using the Random Primers DNA Labeling System (Invitrogen) before hybridization. Sizes of the hybridizing bands were determined relative to 18S and 28S, and found to be in accordance with previous reports. For quantitative assessments, the intensities of the transcription factor signals were normalized to the hybridization intensity from a probe against 18S.
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cells expressing CD11b were positively selected to reduce the contamination by cells, which are at stages of differentiation earlier than the myelocyte. At day 6, cells expressing CD49d were depleted to reduce the amount of cells, which are at stages of differentiation earlier than the metamyelocyte. As shown in Fig. 1B, the expression profile of CD49d in HL60 cells was not affected by the depletion of CD11b positive cells at day 0. Very few cells were CD11b positive in the “day 0” population (1.7%). This number was reduced to 0.8% by depletion. May-Gr¨unwald Giemsa staining of HL60 “day 0 cells” is shown in Fig. 1C, depicting myeloblast-like cells with high nucleus to cytoplasm ratio and almost round nuclei. At day 3, the CD49d level in the mixed HL60 cell culture began to decline, and positive selection of CD11b-expressing cells resulted in further decline of CD49d expressing cells (Fig. 1B). May-Gr¨unwald Giemsa staining showed cells that morphologically resemble neutrophils at the myelocyte–metamyelocyte stage (Fig. 1C). At day 6 of differentiation, the CD49d expression was highly varying, reflecting a mixture of cells at all stages of differentiation. However, after depletion of CD49d-expressing cells only cells with a weak or no CD49d-expression remained. This treatment also removed cells that did not express CD11b, reflecting their stage of immaturity (Fig. 1B). May-Gr¨unwald Giemsa staining showed mainly cells with a lobulised nucleus and only very few cells with round or myelocyte-like nuclei (Fig. 1C). NB4 cells were purified in the same manner as HL60 cells, and demonstrated similar morphology and surface expression-pattern of CD11b and CD49d following MACS purification as observed in HL60 cells (Fig. 1B and C).
3. Results 3.1. In vitro differentiation of HL60 and NB4 cells and purification by immunomagnetic separation To investigate the expression of transcription factors during myelopoiesis in vitro, differentiation of HL60 and NB4 cells was induced by addition of 1.3% DMSO + 10−6 M ATRA and 10−6 M ATRA, respectively. Differentiation was not synchronized, as a mixture of cells of different maturity was observed at all times during in vitro differentiation. Even after 6 days of in vitro differentiation some immature cells could be identified although a large number of cells were morphologically mature. To obtain more homogeneous cell populations, the cells were purified by immunomagnetic separation according to their content of cell surface markers CD11b and CD49d. CD11b is not found on the surface of the myeloblast or promyelocyte, but is expressed at the myelocyte stage and onwards (Fig. 1A) [32]. In contrast, CD49d is present on the surface of early neutrophil precursors, becomes down-regulated at the metamyelocyte stage and is absent from more mature neutrophils (Fig. 1A) [32]. Day 0 cells were depleted for CD11b-expressing cells, giving a cell population depleted for spontaneously differentiated cells. At day 2 (NB4) or 3 (HL60) of in vitro differentiation,
3.2. Expression-pattern of stage-specific markers of neutrophil differentiation To further verify that the three cell populations represent cells of different maturity we purified RNA from the cells for Northern blotting. As seen in Fig. 1D and E, high expression of MPO, an azurophil granule protein whose transcript level peaks in the myeloblast and promyelocyte in vivo [33], was found in HL60 “day 0” cells. MPO expression was very weak in “day 3 cells” and no expression could be detected at day 6. fMLP-receptor (fMLP-R) is a membrane protein found in gelatinase granules, secretory vesicles, and on the cell surface of mature neutrophils. In vivo, the transcript appears at the band cell stage of maturation and remains expressed in mature neutrophils [33]. Here, the fMLP-R transcript was undetectable in HL60 “day 0 cells”, weakly expressed in “day 3 cells”, and highly expressed in “day 6 cells” following depletion (Fig. 1D and E). When RNA was extracted at day 6, before depletion of CD49d-expressing cells, a fivefold lower level of fMLP-R transcript was found (data not shown). This reflects the large amount of cells, which have not reached the stage of maturation where fMLP-R transcription is initiated in the un-purified day-6 cell culture, and underscores the importance of the depletion step. When MPO and
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Fig. 1. In vitro differentiation of HL60 and NB4 cells, and further purification according to cell surface markers. (A) Schematic representation of the surface expression of CD11b and CD49d during in vivo neutrophil differentiation [32]. (B) Flow-cytometric analysis of CD11b and CD49d surface expression during in vitro differentiation of HL60 (left) and NB4 (right) cells. The cells were analyzed day 0 prior to and following depletion of CD11b-expressing cells, day 3 (or 2 for NB4) prior to or following positive selection of CD11b-expressing cells, and day 6 prior to and following depletion of CD49d expressing cells. Blue: antigen expression; white: isotype control. (C) May-Gr¨unwald Giemsa stainings of MACS depleted or selected HL60 and NB4 cells. (D) Northern blot of MPO and fMLP-R in MACS purified in vitro differentiated HL60 and NB4 cells. The blots were hybridized to 18S as a loading control. (E) Relative hybridization intensities of the blots in (D) related to 18S. For each probe the cell population with the strongest hybridization signal was given the value 1, and the other signal intensities related to this. For both cell lines, the results shown are representative for two independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
fMLP-R transcript levels were analyzed in NB4 cells, similar results were obtained, except for a slightly higher expression of MPO in “day 2 cells” than observed in the HL60 “day 3 cells” (Fig. 1D and E). Together, these data demonstrate that three populations of HL60 and NB4 cells have been obtained, which represent three different stages of maturity. As determined by morphology, surface expression of CD11b and CD49d, and mRNA profiles for MPO and fMLP-R, the three populations of purified cells from HL60 and NB4, respectively, were at similar maturation stages. Furthermore, the maturity of the three cell
populations were comparable to the three neutrophil precursor populations isolated from normal human bone marrow in our previous study, where we looked at transcription factor expression during differentiation in vivo [31]. 3.3. Expression profiles of transcription factor mRNAs during in vitro differentiation of HL60 cells RNA was harvested from the three HL60 cell populations and used for Northern blotting, and the expression profiles of 14 different transcription factor mRNAs were
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Fig. 2. Northern blotting of transcription factors in HL60 cells. Northern blots of total RNA from HL60 days 0, 3, and 6. (A–F) Different membranes hybridized with the indicated probes. Each membrane was hybridized to 18S as loading control. The results shown are representative for two independent experiments.
analyzed (Figs. 2 and 3). In general, all the transcription factors previously observed to be expressed during normal neutrophil differentiation could be detected, but showed a more uniform expression-pattern in vitro, rather than the distinct profiles observed in vivo. For Runx1, C/EBP, and c-Fos, the observed mRNA profiles differed widely from the profiles observed in vivo. For Runx1, a high expression was seen throughout in vitro differentiation in contrast to the down-regulation observed in vivo. For C/EBP the highest expression was observed in undifferentiated HL60 cells, which does not correspond to the in vivo situation where the transcript does not appear until the band cell stage of maturation. c-Fos transcripts can be detected at similar levels in all three HL60 populations. In vivo, c-Fos is barely detectable before the band cell stage where the transcript level increases dramatically. C/EBP, C/EBP, C/EBP␦, PU.1, and c-jun had mRNA profiles in HL60 cells with trends similar to the profiles observed in vivo. The down-regulation of C/EBP
transcript in the most mature cells in vivo was not as pronounced in vitro. In HL60 cells, the transcripts for C/EBP, C/EBP␦, and PU.1 were up-regulated during in vitro differentiation but did not increase as much as observed in vivo. The mRNA profiles of GATA-1, c-myb, C/EBP␥, Elf-1, and CCAAT displacement protein (CDP) in HL60 cells were almost identical to the profiles observed in vivo ([31] and Fig. 6A). 3.4. Expression profiles of transcription factor mRNAs during in vitro differentiation of NB4 cells RNA from the three NB4 populations was also analyzed for mRNAs of transcription factors (Figs. 4 and 5). The examined transcription factors were all expressed in the NB4 cells but also in this case were the profiles different from the profiles observed in vivo ([31] and Fig. 6A). Many transcription factors had uniform expression during in vitro differentiation
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Fig. 3. Schematic representation of transcript levels in HL60 cells. Relative hybridization intensities of the blots in Fig. 2 following normalization to 18S. For each probe, the cell population showing maximal expression is given the value 1; the expression levels in the other cell populations are shown relative to this. The results shown are representative for two independent experiments.
of NB4 cells in contrast to the specific profiles in vivo and the overall picture was comparable to that of HL60 cells. The transcription factors whose profiles differed most from the in vivo profiles were GATA-1, C/EBP␥, Runx1, C/EBP␣, C/EBP, and C/EBP. GATA-1, C/EBP␥ and Runx1 transcripts were present at all stages of in vitro differentiation of NB4 cells. This is in sharp contrast to the down-regulation
observed during in vivo differentiation. The transcript level of C/EBP␣ is rapidly down-regulated during in vitro differentiation of NB4 cells, whereas a more steady level was found in vivo. The C/EBP mRNA was rapidly up-regulated during in vitro differentiation of NB4 cells corresponding well to the in vivo situation, but the down-regulation observed in vivo at the later stage of maturation could not be seen in vitro. As
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Fig. 4. Northern blotting of transcription factors in NB4 cells. Northern blot of total RNA from NB4 days 0, 2, and 6. (A–E) Different membranes hybridized with the indicated probes. Each membrane was hybridized to 18S as a loading control. The results shown are representative for two independent experiments.
for HL60 cells, the transcript for C/EBP was only present in the immature NB4 cells in contrast to the presence only in the most mature cells in vivo.
4. Discussion We wished to compare transcription factor mRNA profiles during in vitro differentiation of HL60 and NB4 cells to the pattern observed in vivo. The mRNA profiles previously observed in vivo were very distinct, and steep up- and downregulations of key factors were observed concomitantly with the transition from one cell population to another [31]. Furthermore, the protein profiles were found to match the mRNA profiles in vivo [31]. When in vitro differentiation of HL60 and NB4 cells was induced, it resulted in a mixture of cells with different maturity (Fig. 1). In order to compare changes
in transcription factor mRNA levels during maturation of HL60 and NB4 cells to that seen in neutrophils, the cells were further purified by use of the cell surface markers CD11b and CD49d. This resulted in three populations of cells of progressive maturity where contamination with cells at other stages of maturity was reduced to a level where they would not significantly influence the results. The same transcription factors previously analyzed during in vivo differentiation were analyzed in HL60 and NB4 cells, and transcripts for all, with the exception of GATA-1, were found to be expressed in both cell lines. GATA-1 was only detected in NB4 cells (Figs. 2–5). The profiles are summarised in Fig. 6 where the in vivo profile is shown for comparison. Even though the cells were purified according to cell surface markers in order to reduce contamination of cells with inappropriate maturity, the observed pattern of transcription factor mRNAs during in vitro differentiation of HL60 and NB4 cells did not resemble
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Fig. 5. Schematic representation of transcript levels in NB4 cells. Relative hybridization intensities of the blots in Fig. 4 normalized to 18S. For each probe, the cell population showing maximal expression is given the value 1; the expression levels in the other cell populations are shown relative to this. The results shown are representative for two independent experiments.
the pattern observed in vivo. This indicates a general dysregulation of the transcription factors at the transcript level during in vitro differentiation of HL60 and NB4 presumably resulting in incorrect relative concentrations of the transcription factors. This is an important point since cell lines have been used in many studies as models for transcription factor expression during normal neutrophil differentiation and are widely referred to as the normal expression-patterns [34–40].
Our data show that great care should be exerted when using HL60 and NB4 for the study of transcription factor regulation as exemplified by C/EBP where the same expression profile is seen in the two cell lines and therefore could be interpreted as the “true” expression profile of this transcription factor. This, however, is an erroneous interpretation as a quite different expression profile is seen in vivo (Fig. 6 and [31]).
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Fig. 6. Schematic representation of transcription factor profiles during in vitro differentiation of HL60 and NB4 cells. (A) Distribution of transcription factors during in vivo neutrophil differentiation as previously reported [31]. MB: myeloblast, PM: promyelocyte, MC: myelocyte, MM: metamyelocyte, BC: band cell, and SC: segmented neutrophil cell. (B) Theoretical distribution scheme of the transcription factors during in vitro differentiation of HL60 cells examined in this work based on the data presented in Figs. 2 and 3. (C) Theoretical distribution scheme of the transcription factors during in vitro differentiation of NB4 cells examined in this work based on the data presented in Figs. 4 and 5. Gray profiles have similar trends as the profiles observed in vivo, black profiles differ widely from the in vivo profiles.
Specific granule proteins are not expressed during in vitro differentiation of HL60 and NB4 cell lines [26,25,29]. It is known that C/EBP is essential for transcription of the genes encoding specific granule proteins [14,41,42]. However, as both cell lines express C/EBP mRNA (Figs. 2 and 4) and protein (as reported by others [43,36] and in this study (data not shown)) this indicates that the mere expression of C/EBP is not sufficient to induce their transcription. CDP is a transcriptional repressor, which also is important for regulation of specific granule protein gene expression. In this case CDP binding to the specific granule protein promoters is needed to
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be down-regulated in order to allow their expression [44,41]. Down-regulation of CDP binding to another CDP target promoter, the gp91phox promoter, during in vitro differentiation of HL60 and NB4 cells has been reported [45,41] and expression of gp91phox protein was observed in this study as measured by Nitroblue tetrazolium (NBT) reduction [27] (data not shown). This indicates that repression by CDP is in fact down-regulated during in vitro differentiation of HL60 and NB4 cells. In NB4 cells CDP has been reported to remain attached to the lactoferrin promoter during in vitro differentiation in contrast to the release of the gp91phox promoter [41] but the reason for this difference was not identified. Together, this suggests that CDP and C/EBP are not the only critical factors for transcriptional regulation of the specific granule matrix proteins. C/EBP␣ and PU.1 can also induce expression of specific granule proteins [42,46] but as their transcripts were present during in vitro differentiation of HL60 and NB4 cells, this also cannot explain the lack of specific granule protein expression during in vitro differentiation of HL60 and NB4 cells, although altered posttranscriptional or posttranslational modifications cannot be ruled out. The continued high expression of Runx1 during in vitro differentiation of both HL60 and NB4 cells, in contrast to the observed down-regulation in vivo, is notable, and it could be speculated that Runx1 has a function as a repressor for the specific granule proteins. Runx1 can act both as an activator and as a repressor of transcription, dependent on the cell type and the promoter upon which it acts. This is exemplified by the concomitant activation of CD8 transcription and repression of CD4 transcription by Runx1 in T cells [47]. In conclusion we have found that transcription factor mRNA profiles during in vitro differentiation of HL60 and NB4 cells do not correspond to each other or to the profile observed in vivo. Although HL60 and NB4 cells may be good models for many aspects of neutrophil differentiation, one should carefully consider the usefulness of these cell lines if one wants to study transcription factor regulation during granulopoiesis.
Acknowledgements The expert technical assistance of Inge Kobbernagel is greatly appreciated. The authors wish to thank Pia Klausen for fruitful suggestions to the experiments. This work was supported by grants from The Danish Cancer Society, The Danish Medical Research Council, Copenhagen University Hospital (H:S), and The Danish Foundation for Cancer Research.
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[21] Koschmieder S, Rosenbauer F, Steidl U, Owens BM, Tenen DG. Role of transcription factors C/EBPalpha and PU.1 in normal hematopoiesis and leukemia. Int J Hematol 2005;81:368–77. [22] Dahl R, Walsh JC, Lancki D, Laslo P, Iyer SR, Singh H, et al. Regulation of macrophage and neutrophil cell fates by the PU.1:C/EBPalpha ratio and granulocyte colony-stimulating factor. Nat Immunol 2003;4:1029–36. [23] Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci 1978;75:2458–62. [24] Breitman TR, He RY. Combinations of retinoic acid with either sodium butyrate, dimethyl sulfoxide, or hexamethylene bisacetamide synergistically induce differentiation of the human myeloid leukemia cell line HL60. Cancer Res 1990;50:6268–73. [25] Le Cabec V, Cowland JB, Calafat J, Borregaard N. Targeting of proteins to granule subsets is determined by timing and not by sorting: the specific granule protein NGAL is localized to azurophil granules when expressed in HL-60 cells. Proc Natl Acad Sci USA 1996;93:6454–7. [26] Johnston JJ, Rintels P, Chung J, Sather J, Benz Jr EJ, Berliner N. Lactoferrin gene promoter: structural integrity and nonexpression in HL60 cells. Blood 1992;79:2998–3006. [27] Le Cabec V, Calafat J, Borregaard N. Sorting of the specific granule protein, NGAL, during granulocytic maturation of HL-60 cells. Blood 1997;89:2113–21. [28] Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, Berger R. NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3). Blood 1991;77:1080–6. [29] Khanna-Gupta A, Kolibaba K, Zibello TA, Berliner N. NB4 cells show bilineage potential and an aberrant pattern of neutrophil secondary granule protein gene expression. Blood 1994;84:294–302. [30] Gregoire C, Welch H, Astarie-Dequeker C, Maridonneau-Parini I. Expression of azurophil and specific granule proteins during differentiation of NB4 cells in neutrophils. J Cell Physiol 1998;175:203–10. [31] Bjerregaard MD, Jurlander J, Klausen P, Borregaard N, Cowland JB. The in vivo profile of transcription factors during neutrophil differentiation in human bone marrow. Blood 2003;101:4322–32. [32] Lund JF, Terstappen LW. Differential surface expression of cell adhesion molecules during granulocyte maturation. J Leukoc Biol 1993;54:47–55. [33] Cowland JB, Borregaard N. The individual regulation of granule protein mRNA levels during neutrophil maturation explains the heterogeneity of neutrophil granules. J Leukoc Biol 1999;66:989–95. [34] Chih DY, Chumakov AM, Park DJ, Silla AG, Koeffler HP. Modulation of mRNA expression of a novel human myeloid-selective CCAAT/enhancer binding protein gene (C/EBP epsilon). Blood 1997;90:2987–94. [35] Yamanaka R, Kim G-D, Radomska HS, Lekstrom-Himes J, Smith LT, Antonson P, et al. CCAAT/enhancer binding protein is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing. Proc Natl Acad Sci 1997;94:6462–7. [36] Park DJ, Chumakov AM, Vuong PT, Chih DY, Gombart AF, Miller WHJ, et al. CCAAT/enhancer binding protein epsilon is a potential retinoid target gene in acute promyelocytic leukemia treatment. J Clin Invest 1999;103:1399–408. [37] Hromas R, Collins SJ, Hickstein D, Raskind W, Deaven LL, O’Hara P, et al. A retinoic acid-responsive human zinc finger gene, MZF-1, preferentially expressed in myeloid cells. J Biol Chem 1991;266:14183–7. [38] Scott LM, Civin CI, Rorth P, Friedman AD. A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells. Blood 1992;80:1725–35. [39] Radomska HS, Huettner CS, Zhang P, Cheng T, Scadden DT, Tenen DG. CCAAT/enhancer binding protein alpha is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors. Mol Cell Biol 1998;18:4301–14.
M.B. Pass et al. / Leukemia Research 31 (2007) 827–837 [40] Gery S, Park DJ, Vuong PT, Chih DY, Lemp N, Koeffler HP. Retinoic acid regulates C/EBP homologous protein expression (CHOP), which negatively regulates myeloid target genes. Blood 2004;104: 3911–7. [41] Khanna-Gupta A, Zibello T, Sun H, Gaines P, Berliner N. Chromatin immunoprecipitation (ChIP) studies indicate a role for CCAAT enhancer binding proteins alpha and epsilon (C/EBP alpha and C/EBP epsilon) and CDP/cut in myeloid maturation-induced lactoferrin gene expression. Blood 2003;101:3460–8. [42] Gombart AF, Kwok SH, Anderson KL, Yamaguchi Y, Torbett BE, Koeffler HP. Regulation of neutrophil and eosinophil secondary granule gene expression by transcription factors C/EBP epsilon and PU.1. Blood 2003;101:3265–73. [43] Verbeek W, Gombart AF, Chumakov AM, Muller C, Friedman AD, Koeffler HP. C/EBPepsilon directly interacts with the DNA binding
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domain of c-myb and cooperatively activates transcription of myeloid promoters. Blood 1999;93:3327–37. Khanna-Gupta A, Zibello T, Kolla S, Neufeld EJ, Berliner N. CCAAT displacement protein (CDP/cut) recognizes a silencer element within the lactoferrin gene promoter. Blood 1997;90:2784–95. Lievens PM, Donady JJ, Tufarelli C, Neufeld EJ. Repressor activity of CCAAT displacement protein in HL-60 myeloid leukemia cells. J Biol Chem 1995;270:12745–50. Khanna-Gupta A, Zibello T, Simkevich C, Rosmarin AG, Berliner N. Sp1 and C/EBP are necessary to activate the lactoferrin gene promoter during myeloid differentiation. Blood 2000;95:3734–41. Taniuchi I, Osato M, Egawa T, Sunshine MJ, Bae SC, Komori T, et al. Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development. Cell 2002;111:621–33.
Derangement of transcription factor profiles during in vitro differentiation of HL60 and NB4 cells Malene Bjerregaard Pass, Niels Borregaard, Jack Bernard Cowland ∗ The Granulocyte Research Laboratory, Department of Hematology 93.2.2, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Received 23 June 2006; received in revised form 23 June 2006; accepted 16 July 2006 Available online 30 August 2006
Abstract Sequential up- and down-regulation of a handful of critical transcription factors is required for proper neutrophil differentiation. Malfunction of transcription factors may lead to diseases such as acute myeloid leukemia (AML) and specific granule deficiency. In order to understand the molecular background for normal and malignant granulopoiesis, a good model system is required that faithfully mimics the in vivo transcription factor expression profiles. The two human leukemic cell lines HL60 and NB4 have been widely used as model cell lines for these purposes. Differentiation of HL60 and NB4 cells resulted in asynchronous differentiation to morphologically mature neutrophils over a period of 5–7 days. To obtain cell populations of more even maturity, cells at different stages of in vitro differentiation were purified by immunomagnetic isolation. This resulted in three cell populations that could be classified as promyelocytes, myelocytes/metamyelocytes, and mature neutrophils, respectively. Comparison of transcription factor mRNA profiles from these cell populations with those previously seen in normal human bone marrow, demonstrated that although all of the 14 transcription factors described in vivo, could be detected during in vitro differentiation, vast differences in their expression profiles was observed. These data illustrate the limitations of cell lines as models for normal granulopoiesis. © 2006 Elsevier Ltd. All rights reserved. Keywords: HL60; NB4; AML; Granulopoiesis; Transcription factors; Granule proteins; C/EBP; PU.1
1. Introduction Formation of the neutrophil granulocyte takes place in the bone marrow. A number of transcription factors play a critical role during this process and mutations of transcription factor genes may lead to aberrant maturation of neutrophils. Chromosomal translocations involving Runx1 (also called AML1) [1], and mutations or deregulation of C/EBP␣ [2–4] is frequently observed in acute myeloid leukemia (AML) patients. Furthermore, mutations of PU.1 have in some cases been associated with AML [5] and disruption of C/EBP has been observed in patients with specific granule deficiency [6]. Gene disruption studies in mice have also identified several transcription factors essential for neutrophil devel∗
Corresponding author. Tel.: +45 35 45 48 88; fax: +45 35 45 67 27. E-mail address: [email protected] (J.B. Cowland).
0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2006.07.019
opment. Lack of definitive hematopoiesis is observed in Runx1−/− or c-myb−/− fetuses, demonstrating their importance for all hematopoietic lineages, including neutrophils [7–9]. Mice deficient in C/EBP␣ completely lack mature neutrophils and eosinophils, whereas all other hematopoietic lineages are present, and function normally [10]. Immature myeloid precursor cells are found in the blood of C/EBP␣ knock-out mice, demonstrating that C/EBP␣ is essential for neutrophil differentiation [10]. Conditional knock-outs of C/EBP␣ have demonstrated that C/EBP␣ is essential for the transition from the common myeloid progenitor (CMP) to the granulocyte/macrophage restricted progenitor (GMP), but not for GMPs to terminally differentiate to mature neutrophils [11]. C/EBP-deficient mice also fail to develop fully mature neutrophils and eosinophils, only atypical hyposegmented neutrophils were present [12]. Transcripts for azurophil granule proteins were detected in the bone marrow, whereas an
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almost complete lack of transcripts for specific granule proteins was noted [12–14]. Mice deficient in PU.1 also generate neutrophils with a block in differentiation [15–17]. Conditional knock-outs have demonstrated that PU.1 is essential for the generation of CMPs, and for the terminal maturation of GMPs to mature neutrophils [18,19]. Analysis of knock-out mice is valuable if one wish to demonstrate the requirement of a given transcription factor for myeloid differentiation. However, due to the inherent property of knock-out studies this method only allows one to determine the earliest point of differentiation where the transcription factor in question is essential. Furthermore, as it is becoming evident that not only the presence of a transcription factor, but also its overall concentration [20,21] – as well as its concentration relative to that of other factor transcription factors [22] – is important for lineage decision and differentiation, model systems to investigate these issues are also required. Cellular systems to study transcription factor regulation during myeloid differentiation in vitro may therefore be of great value provided the models faithfully mimick the in vivo profile of transcription factor expression. Two human leukemic cell lines, HL60 and NB4, are widely used as models for neutrophil differentiation. The HL60 cell line was established in 1977 from a patient suffering from AML, FAB M2 [23]. The cells grow continuously as myeloblasts and can differentiate to morphologically mature looking neutrophils after addition of all-trans retinoic acid (ATRA) and dimethyl sulfoxide (DMSO) [24]. The differentiation is not complete since specific granules and gelatinase granules are not formed and transcripts for matrix proteins contained in these granules are not detectable [25,26]. Membrane proteins of these granules are synthesized but are routed to the plasma membrane [27]. Constitutive expression of the specific granule protein NGAL in HL60 cells results in an accumulation of NGAL in the azurophil granules when the cells are at the myeloblast stage. However, when differentiation of HL60 cells is induced, newly synthesized NGAL is routed extracellularly and does not accumulate in granules, reflecting the inability of more mature HL60 cells to synthesize granules and retain granule proteins [25,27]. The NB4 cell line was established in 1991 from a patient suffering from acute promyelocyte leukemia (APL) having the t(15;17) translocation [28]. Morphologically, the cells are characterized as promyelocytes and can be stimulated to differentiate to neutrophils by ATRA. Like HL60 cells, specific and gelatinase granules are not formed during in vitro differentiation of NB4 cells and mRNA for specific granule matrix proteins cannot be detected [29]. Also like in HL60 cells, membrane proteins of specific and gelatinase granules are synthesized when neutrophil maturation is induced and routed to the plasma membrane [30]. We have previously used a method for separation of neutrophil precursors from human bone marrow into three populations of different maturity. These are myeloblasts (MB) + promyelocytes (PM), myelocytes (MC) + metamyelocytes (MM), and band cells (BC) + seg-
mented neutrophil cells (SC), respectively. By analyzing these populations by Northern and Western blotting, a highly individualized expression of 14 transcription factors important for neutrophil differentiation was demonstrated during in vivo granulopoiesis [31]. The purpose of this study was to correlate the expressionpattern of transcription factor mRNAs in HL60 and NB4 cells during in vitro differentiation, to the in vivo pattern referred to above and to determine whether these two leukemic cell lines may be used as model systems for transcription factor regulation during granulopoiesis.
2. Materials and methods 2.1. In vitro differentiation of HL60 and NB4 cells HL60 cells (CCL-240) were obtained from American Type Culture Collection (ATCC) and NB4 cells were generously provided by Dr. M. Lanotte [28]. The cells were cultured in RPMI 1640 with glutamax-1 (Invitrogen, San Diego, CA, USA) supplemented with 10% fetal calf serum (FCS) (Invitrogen), 100 units/ml penicillin (Invitrogen), and 100 g/ml streptomycin (Invitrogen). In vitro differentiation of HL60 was induced by addition of 10−6 M ATRA (Sigma–Aldrich, St. Louis, MO, USA) and 1.3% DMSO and of NB4 by addition of 10−6 M ATRA. 2.2. Purification of cells by immunomagnetic isolation Exponentially growing NB4 or HL60 cells were depleted for spontaneously differentiated cells expressing CD11b by addition of anti CD11b antibody (BD Pharmingen, San Diego, CA, USA) followed by MACS beads, anti-IgG1 (10 l/107 cells) (Miltenyi Biotech, Bergisch Gladbach, Germany), and depleted using an AS column (Miltenyi Biotech). We refer to these depleted populations as “day 0 cells”. At day 2 (NB4) or day 3 (HL60) of the in vitro differentiation, cells expressing CD11b were positively selected. The cells were incubated with anti CD11b antibody followed by MACS beads, anti-IgG1 (10 l/107 cells), and positively selected on a BS column (Miletenyi Biotech). We refer to these positive selected populations as “day 2 cells” and “day 3 cells”. At day 6 of in vitro differentiation, cells still expressing CD49d were depleted. The cells were incubated with anti CD49d antibody (BD Pharmingen) followed by MACS beads, antiIgG1 (10 l/107 cells), and depleted on a BS column. We refer to these depleted populations as “day 6 cells”. In vitro differentiation and immunomagnetic isolation was performed twice for both cell lines with very similar results—the result of one experiment is shown. 2.3. Flow-cytometric analysis For flow-cytometric analysis the cells were stained with anti CD11b-PE (mouse IgG1 , BD Pharmingen), CD49d-PE
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(mouse IgG1 , BD Pharmingen), or isotype control. Cells were incubated with antibody for 15 min at room temperature for labeling, washed twice in 0.5% BSA in PBS, and fixed in 1% paraformaldehyde in PBS. The cells were analyzed on a FACScan instrument (Becton Dickinson, San Jose, CA, USA). Data was analyzed using the CellQuest software. 2.4. Northern blotting Total RNA was isolated with Trizol (Invitrogen). The RNA was ethanol-precipitated and resuspended in 0.1 mM EDTA. For Northern blotting, 5 g of RNA was run on a 1% agarose-gel and transferred to a Hybond-N membrane (GE Healthcare, Chalfont ST. Giles, United Kingdom) as described [31]. Filters were pre-hybridized for 1–2 h at 42 ◦ C in 6 ml ULTRAhyb (Ambion, Austin, TX, USA) and hybridized overnight at 42 ◦ C after addition of further 4 ml containing the 32 P-labeled probe and sheared salmon sperm DNA (10 g/ml). The membranes were washed as described [31] and developed by a Fuji BAS2500 PhosphorImager. Membranes were stripped by boiling in 0.1% SDS before re-hybridization. The probes are described previously [31]. They were radiolabeled with [␣-32 P] dCTP using the Random Primers DNA Labeling System (Invitrogen) before hybridization. Sizes of the hybridizing bands were determined relative to 18S and 28S, and found to be in accordance with previous reports. For quantitative assessments, the intensities of the transcription factor signals were normalized to the hybridization intensity from a probe against 18S.
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cells expressing CD11b were positively selected to reduce the contamination by cells, which are at stages of differentiation earlier than the myelocyte. At day 6, cells expressing CD49d were depleted to reduce the amount of cells, which are at stages of differentiation earlier than the metamyelocyte. As shown in Fig. 1B, the expression profile of CD49d in HL60 cells was not affected by the depletion of CD11b positive cells at day 0. Very few cells were CD11b positive in the “day 0” population (1.7%). This number was reduced to 0.8% by depletion. May-Gr¨unwald Giemsa staining of HL60 “day 0 cells” is shown in Fig. 1C, depicting myeloblast-like cells with high nucleus to cytoplasm ratio and almost round nuclei. At day 3, the CD49d level in the mixed HL60 cell culture began to decline, and positive selection of CD11b-expressing cells resulted in further decline of CD49d expressing cells (Fig. 1B). May-Gr¨unwald Giemsa staining showed cells that morphologically resemble neutrophils at the myelocyte–metamyelocyte stage (Fig. 1C). At day 6 of differentiation, the CD49d expression was highly varying, reflecting a mixture of cells at all stages of differentiation. However, after depletion of CD49d-expressing cells only cells with a weak or no CD49d-expression remained. This treatment also removed cells that did not express CD11b, reflecting their stage of immaturity (Fig. 1B). May-Gr¨unwald Giemsa staining showed mainly cells with a lobulised nucleus and only very few cells with round or myelocyte-like nuclei (Fig. 1C). NB4 cells were purified in the same manner as HL60 cells, and demonstrated similar morphology and surface expression-pattern of CD11b and CD49d following MACS purification as observed in HL60 cells (Fig. 1B and C).
3. Results 3.1. In vitro differentiation of HL60 and NB4 cells and purification by immunomagnetic separation To investigate the expression of transcription factors during myelopoiesis in vitro, differentiation of HL60 and NB4 cells was induced by addition of 1.3% DMSO + 10−6 M ATRA and 10−6 M ATRA, respectively. Differentiation was not synchronized, as a mixture of cells of different maturity was observed at all times during in vitro differentiation. Even after 6 days of in vitro differentiation some immature cells could be identified although a large number of cells were morphologically mature. To obtain more homogeneous cell populations, the cells were purified by immunomagnetic separation according to their content of cell surface markers CD11b and CD49d. CD11b is not found on the surface of the myeloblast or promyelocyte, but is expressed at the myelocyte stage and onwards (Fig. 1A) [32]. In contrast, CD49d is present on the surface of early neutrophil precursors, becomes down-regulated at the metamyelocyte stage and is absent from more mature neutrophils (Fig. 1A) [32]. Day 0 cells were depleted for CD11b-expressing cells, giving a cell population depleted for spontaneously differentiated cells. At day 2 (NB4) or 3 (HL60) of in vitro differentiation,
3.2. Expression-pattern of stage-specific markers of neutrophil differentiation To further verify that the three cell populations represent cells of different maturity we purified RNA from the cells for Northern blotting. As seen in Fig. 1D and E, high expression of MPO, an azurophil granule protein whose transcript level peaks in the myeloblast and promyelocyte in vivo [33], was found in HL60 “day 0” cells. MPO expression was very weak in “day 3 cells” and no expression could be detected at day 6. fMLP-receptor (fMLP-R) is a membrane protein found in gelatinase granules, secretory vesicles, and on the cell surface of mature neutrophils. In vivo, the transcript appears at the band cell stage of maturation and remains expressed in mature neutrophils [33]. Here, the fMLP-R transcript was undetectable in HL60 “day 0 cells”, weakly expressed in “day 3 cells”, and highly expressed in “day 6 cells” following depletion (Fig. 1D and E). When RNA was extracted at day 6, before depletion of CD49d-expressing cells, a fivefold lower level of fMLP-R transcript was found (data not shown). This reflects the large amount of cells, which have not reached the stage of maturation where fMLP-R transcription is initiated in the un-purified day-6 cell culture, and underscores the importance of the depletion step. When MPO and
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Fig. 1. In vitro differentiation of HL60 and NB4 cells, and further purification according to cell surface markers. (A) Schematic representation of the surface expression of CD11b and CD49d during in vivo neutrophil differentiation [32]. (B) Flow-cytometric analysis of CD11b and CD49d surface expression during in vitro differentiation of HL60 (left) and NB4 (right) cells. The cells were analyzed day 0 prior to and following depletion of CD11b-expressing cells, day 3 (or 2 for NB4) prior to or following positive selection of CD11b-expressing cells, and day 6 prior to and following depletion of CD49d expressing cells. Blue: antigen expression; white: isotype control. (C) May-Gr¨unwald Giemsa stainings of MACS depleted or selected HL60 and NB4 cells. (D) Northern blot of MPO and fMLP-R in MACS purified in vitro differentiated HL60 and NB4 cells. The blots were hybridized to 18S as a loading control. (E) Relative hybridization intensities of the blots in (D) related to 18S. For each probe the cell population with the strongest hybridization signal was given the value 1, and the other signal intensities related to this. For both cell lines, the results shown are representative for two independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
fMLP-R transcript levels were analyzed in NB4 cells, similar results were obtained, except for a slightly higher expression of MPO in “day 2 cells” than observed in the HL60 “day 3 cells” (Fig. 1D and E). Together, these data demonstrate that three populations of HL60 and NB4 cells have been obtained, which represent three different stages of maturity. As determined by morphology, surface expression of CD11b and CD49d, and mRNA profiles for MPO and fMLP-R, the three populations of purified cells from HL60 and NB4, respectively, were at similar maturation stages. Furthermore, the maturity of the three cell
populations were comparable to the three neutrophil precursor populations isolated from normal human bone marrow in our previous study, where we looked at transcription factor expression during differentiation in vivo [31]. 3.3. Expression profiles of transcription factor mRNAs during in vitro differentiation of HL60 cells RNA was harvested from the three HL60 cell populations and used for Northern blotting, and the expression profiles of 14 different transcription factor mRNAs were
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Fig. 2. Northern blotting of transcription factors in HL60 cells. Northern blots of total RNA from HL60 days 0, 3, and 6. (A–F) Different membranes hybridized with the indicated probes. Each membrane was hybridized to 18S as loading control. The results shown are representative for two independent experiments.
analyzed (Figs. 2 and 3). In general, all the transcription factors previously observed to be expressed during normal neutrophil differentiation could be detected, but showed a more uniform expression-pattern in vitro, rather than the distinct profiles observed in vivo. For Runx1, C/EBP, and c-Fos, the observed mRNA profiles differed widely from the profiles observed in vivo. For Runx1, a high expression was seen throughout in vitro differentiation in contrast to the down-regulation observed in vivo. For C/EBP the highest expression was observed in undifferentiated HL60 cells, which does not correspond to the in vivo situation where the transcript does not appear until the band cell stage of maturation. c-Fos transcripts can be detected at similar levels in all three HL60 populations. In vivo, c-Fos is barely detectable before the band cell stage where the transcript level increases dramatically. C/EBP, C/EBP, C/EBP␦, PU.1, and c-jun had mRNA profiles in HL60 cells with trends similar to the profiles observed in vivo. The down-regulation of C/EBP
transcript in the most mature cells in vivo was not as pronounced in vitro. In HL60 cells, the transcripts for C/EBP, C/EBP␦, and PU.1 were up-regulated during in vitro differentiation but did not increase as much as observed in vivo. The mRNA profiles of GATA-1, c-myb, C/EBP␥, Elf-1, and CCAAT displacement protein (CDP) in HL60 cells were almost identical to the profiles observed in vivo ([31] and Fig. 6A). 3.4. Expression profiles of transcription factor mRNAs during in vitro differentiation of NB4 cells RNA from the three NB4 populations was also analyzed for mRNAs of transcription factors (Figs. 4 and 5). The examined transcription factors were all expressed in the NB4 cells but also in this case were the profiles different from the profiles observed in vivo ([31] and Fig. 6A). Many transcription factors had uniform expression during in vitro differentiation
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Fig. 3. Schematic representation of transcript levels in HL60 cells. Relative hybridization intensities of the blots in Fig. 2 following normalization to 18S. For each probe, the cell population showing maximal expression is given the value 1; the expression levels in the other cell populations are shown relative to this. The results shown are representative for two independent experiments.
of NB4 cells in contrast to the specific profiles in vivo and the overall picture was comparable to that of HL60 cells. The transcription factors whose profiles differed most from the in vivo profiles were GATA-1, C/EBP␥, Runx1, C/EBP␣, C/EBP, and C/EBP. GATA-1, C/EBP␥ and Runx1 transcripts were present at all stages of in vitro differentiation of NB4 cells. This is in sharp contrast to the down-regulation
observed during in vivo differentiation. The transcript level of C/EBP␣ is rapidly down-regulated during in vitro differentiation of NB4 cells, whereas a more steady level was found in vivo. The C/EBP mRNA was rapidly up-regulated during in vitro differentiation of NB4 cells corresponding well to the in vivo situation, but the down-regulation observed in vivo at the later stage of maturation could not be seen in vitro. As
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Fig. 4. Northern blotting of transcription factors in NB4 cells. Northern blot of total RNA from NB4 days 0, 2, and 6. (A–E) Different membranes hybridized with the indicated probes. Each membrane was hybridized to 18S as a loading control. The results shown are representative for two independent experiments.
for HL60 cells, the transcript for C/EBP was only present in the immature NB4 cells in contrast to the presence only in the most mature cells in vivo.
4. Discussion We wished to compare transcription factor mRNA profiles during in vitro differentiation of HL60 and NB4 cells to the pattern observed in vivo. The mRNA profiles previously observed in vivo were very distinct, and steep up- and downregulations of key factors were observed concomitantly with the transition from one cell population to another [31]. Furthermore, the protein profiles were found to match the mRNA profiles in vivo [31]. When in vitro differentiation of HL60 and NB4 cells was induced, it resulted in a mixture of cells with different maturity (Fig. 1). In order to compare changes
in transcription factor mRNA levels during maturation of HL60 and NB4 cells to that seen in neutrophils, the cells were further purified by use of the cell surface markers CD11b and CD49d. This resulted in three populations of cells of progressive maturity where contamination with cells at other stages of maturity was reduced to a level where they would not significantly influence the results. The same transcription factors previously analyzed during in vivo differentiation were analyzed in HL60 and NB4 cells, and transcripts for all, with the exception of GATA-1, were found to be expressed in both cell lines. GATA-1 was only detected in NB4 cells (Figs. 2–5). The profiles are summarised in Fig. 6 where the in vivo profile is shown for comparison. Even though the cells were purified according to cell surface markers in order to reduce contamination of cells with inappropriate maturity, the observed pattern of transcription factor mRNAs during in vitro differentiation of HL60 and NB4 cells did not resemble
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M.B. Pass et al. / Leukemia Research 31 (2007) 827–837
Fig. 5. Schematic representation of transcript levels in NB4 cells. Relative hybridization intensities of the blots in Fig. 4 normalized to 18S. For each probe, the cell population showing maximal expression is given the value 1; the expression levels in the other cell populations are shown relative to this. The results shown are representative for two independent experiments.
the pattern observed in vivo. This indicates a general dysregulation of the transcription factors at the transcript level during in vitro differentiation of HL60 and NB4 presumably resulting in incorrect relative concentrations of the transcription factors. This is an important point since cell lines have been used in many studies as models for transcription factor expression during normal neutrophil differentiation and are widely referred to as the normal expression-patterns [34–40].
Our data show that great care should be exerted when using HL60 and NB4 for the study of transcription factor regulation as exemplified by C/EBP where the same expression profile is seen in the two cell lines and therefore could be interpreted as the “true” expression profile of this transcription factor. This, however, is an erroneous interpretation as a quite different expression profile is seen in vivo (Fig. 6 and [31]).
M.B. Pass et al. / Leukemia Research 31 (2007) 827–837
Fig. 6. Schematic representation of transcription factor profiles during in vitro differentiation of HL60 and NB4 cells. (A) Distribution of transcription factors during in vivo neutrophil differentiation as previously reported [31]. MB: myeloblast, PM: promyelocyte, MC: myelocyte, MM: metamyelocyte, BC: band cell, and SC: segmented neutrophil cell. (B) Theoretical distribution scheme of the transcription factors during in vitro differentiation of HL60 cells examined in this work based on the data presented in Figs. 2 and 3. (C) Theoretical distribution scheme of the transcription factors during in vitro differentiation of NB4 cells examined in this work based on the data presented in Figs. 4 and 5. Gray profiles have similar trends as the profiles observed in vivo, black profiles differ widely from the in vivo profiles.
Specific granule proteins are not expressed during in vitro differentiation of HL60 and NB4 cell lines [26,25,29]. It is known that C/EBP is essential for transcription of the genes encoding specific granule proteins [14,41,42]. However, as both cell lines express C/EBP mRNA (Figs. 2 and 4) and protein (as reported by others [43,36] and in this study (data not shown)) this indicates that the mere expression of C/EBP is not sufficient to induce their transcription. CDP is a transcriptional repressor, which also is important for regulation of specific granule protein gene expression. In this case CDP binding to the specific granule protein promoters is needed to
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be down-regulated in order to allow their expression [44,41]. Down-regulation of CDP binding to another CDP target promoter, the gp91phox promoter, during in vitro differentiation of HL60 and NB4 cells has been reported [45,41] and expression of gp91phox protein was observed in this study as measured by Nitroblue tetrazolium (NBT) reduction [27] (data not shown). This indicates that repression by CDP is in fact down-regulated during in vitro differentiation of HL60 and NB4 cells. In NB4 cells CDP has been reported to remain attached to the lactoferrin promoter during in vitro differentiation in contrast to the release of the gp91phox promoter [41] but the reason for this difference was not identified. Together, this suggests that CDP and C/EBP are not the only critical factors for transcriptional regulation of the specific granule matrix proteins. C/EBP␣ and PU.1 can also induce expression of specific granule proteins [42,46] but as their transcripts were present during in vitro differentiation of HL60 and NB4 cells, this also cannot explain the lack of specific granule protein expression during in vitro differentiation of HL60 and NB4 cells, although altered posttranscriptional or posttranslational modifications cannot be ruled out. The continued high expression of Runx1 during in vitro differentiation of both HL60 and NB4 cells, in contrast to the observed down-regulation in vivo, is notable, and it could be speculated that Runx1 has a function as a repressor for the specific granule proteins. Runx1 can act both as an activator and as a repressor of transcription, dependent on the cell type and the promoter upon which it acts. This is exemplified by the concomitant activation of CD8 transcription and repression of CD4 transcription by Runx1 in T cells [47]. In conclusion we have found that transcription factor mRNA profiles during in vitro differentiation of HL60 and NB4 cells do not correspond to each other or to the profile observed in vivo. Although HL60 and NB4 cells may be good models for many aspects of neutrophil differentiation, one should carefully consider the usefulness of these cell lines if one wants to study transcription factor regulation during granulopoiesis.
Acknowledgements The expert technical assistance of Inge Kobbernagel is greatly appreciated. The authors wish to thank Pia Klausen for fruitful suggestions to the experiments. This work was supported by grants from The Danish Cancer Society, The Danish Medical Research Council, Copenhagen University Hospital (H:S), and The Danish Foundation for Cancer Research.
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