Two erythroid markers, acetylcholinesterase and hemoglobin, can be reversibly induced in the K-562 cell line after sodium butyrate treatment. In the present ...
(CANCER RESEARCH 46. 6327-6332.
December 1986]
Terminal Erythroid Differentiation in the K-562 Cell Line by 1-ß-D-Arabinofuranosylcytosine:Accompaniment by c-myc Messenger RNA Decrease1 G. L. Bianchi Scarrà ,2 M. Romani, D. A. Coviello, C. Garre, R. Ravazzolo, G. Vidali, and F. Ajinar Department of Biology, I.S.M.I., University of Genova ¡G.L. B. S., D. A. C., C. G., R. R., F. A.], and Laboratory of Molecular Biology, Istituto Nazionale per la Ricerca sul Cancro [M. R., G. y.], 16132 Genova, Italy
ABSTRACT Two erythroid markers, acetylcholinesterase and hemoglobin, can be reversibly induced in the K-562 cell line after sodium butyrate treatment. In the present paper we show that l-/3-D-arabinofuranosylcytosine (araC), induces the coordinate, irreversible expression of these two erythroid markers. This induction occurs at an ara-C concentration (0.05 HIM)that results in K-562 cytostasis and is accompanied by deep morphological changes of cells. The differentiated phenotype is independent of the K562 cell clone used |K-562, K-562 (S), K-562 (S)P| and is associated with the loss of cell renewal capacity. Continuous presence of the inducer is not necessary to achieve terminal differentiation. In contrast to what is seen for other inducers (sodium butyrate and hemin), one of the early effects of ara-C treatment is the marked decrease of c-myc mRNA expression after the first 4 hours of induction, whereas Vro.v and histone 4 expression remain constant during the first 48 h. Our results suggest that ara-C treatment can irreversibly activate the erythroid differentiative program of K-562 cells.
INTRODUCTION Cell line K-562 is a widely used model for erythroid cell differentiation because of the constitutive presence of several erythroid markers: Hb,3 i-antigen, membrane-bound AChE, and others. Many agents have been shown to induce hemoglobin synthesis (1) and we have previously reported that, after sodium butyrate treatment, this cell line also expresses increased levels of AChE in parallel with increased Hb synthesis (2, 3). Sodium butyrate, however, does not trigger the terminal differentiation of K-562 cells. The removal of the chemical is, in fact, followed by the rapid reversal of the differentiated phenotype. Another strong inducer of Hb synthesis, hemin, fails to promote the coordinate expression of AChE and Hb and to commit to terminal differentiation (3). In several biological systems the differentiated condition is associated with the dramatic decrease of c-myc expression (46). The role of this oncogene in cell proliferation (7) and differentiation (4-6), although not completely clear, appears to be extremely important and related to the early events following the irreversible commitment to terminal differentiation. It was recently reported that ara-C promotes the irreversible induction of Hb synthesis in the K-562 cell line (8). This induction, associated with the loss of cell renewal capacity, is a finding consistent with terminal cell differentiation (8). In this study we have examined several parameters of differ entiation in ara-C-treated K-562 cells. Our results indicate that Received 12/13/85: revised 8/7/86; accepted 8/12/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advenisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by CNR grants "Progetto Finalizzato Oncologia" N. 17/83.00711.96 and 84.00772.44 and "Progetto Finalizzato Ingegneria Ge netica" N. 83.0102651: Ministero Publica Istruzione (Grant 40% National Proj ect). 2 To whom requests for reprints should be addressed at Department of Biology. I.S.M.I., University of Genova, Viale Benedetto XV. 6-16132 Genova, Italy. 3The abbreviations used are: Hb, hemoglobin; AChE, acetylcholinesterase (EC 3.1.1.7): ara-C, 1-fÃ--D-arabinofuranosylcytosine; FCS, fetal calf serum.
this compound can induce the coordinate expression of two erythroid markers, AChE and Hb, and that this mode of induc tion is independent of drug removal. We also analyzed c-myc mRNA expression during cell differentiation induced either by ara-C or by hemin and sodium butyrate. These are the most commonly used inducing agents which are unable to commit to terminal differentiation. MATERIALS
AND METHODS
Cell Lines and Cultures. K-562 and K-562 (S) cell lines were kindly provided by Dr. M. Gianni (University of Milan) and by Dr. R. Gambari (University of Ferrara); K-562 (S)P, a subclone from K-562 (S) char acterized in our laboratory, has been described (9). All cell lines were grown in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS, transferrin (1 /ig/ml), penicillin (100 units/ml), and streptomycin (0.1 mg/ml) at 37°Cin a humidified atmosphere with 5% CO2. Cells at a concentration of 2-3 x 105/rnl were induced with 0.05 miviara-C, 0.05 mM hemin, and 1.25 HIMsodium butyrate as previously described (3). Tissue culture media were obtained from Flow Laboratories, Milan, Italy; inducers were purchased from Sigma Chemical Co., St. Louis, MO. Growth rate and cell viability, as determined by the trypan blue dye exclusion test, were monitored daily in all experiments. The exper iments were conducted using the K-562 (S)P subclone unless otherwise indicated. Cultures in semisolid media for clonogenic cells quantitation were performed in «-mediumcontaining 0.33% agar and 10% FCS at a cell concentration of 3 x 103/ml. Colonies that contained at least 50 cells were scored after 7 days. In some experiments cells exposed to ara-C and their corresponding controls were washed, resuspended, and grown in drug-free medium at the same cell concentration. "Quiescent" cells were obtained by washing the cells and resuspending them in 0.2% FCS. Changes in DNA, RNA, and protein synthesis during ara-C treatment were determined by the incorporation of 1 /¿Ciof [3H]thymidine, 1 pd of [3H]uridine, and 1 j»Ciof [3H]leucine into 1-ml aliquots of cells (2.5 x 105/sample point) for 1 h as described by Kelly et al. (7). Hemoglobin and Acetylcholinesterase Determination. Hemoglobin de termination in cells grown in suspension or in cytocentrifuged cell smears was performed as described in Refs. 10 and 11, respectively. Percentage of benzidine-positive cells was scored by counting more than 300 cells. Cells were solubilized (1:4; v/v) with lysis buffer (10 mM citrate phosphate buffer, pH 7.0 containing 1.0% Triton X-100), the lysate was cleared by centrifugation at 40,000 x g for 30 min at 4°C, and the supernatant was used for both Hb and AChE assay. Hemoglobin was determined by spectrophotometric assay (12) and AChE activity was assayed according to the method of Ellman et al. ( 13). This reaction measures the formation of a yellow compound between 5,5-dithiobis2-nitrobenzoic acid (0.62 mM in the reaction mixture) and the thiocholine, which derives from the splitting of acetylthiocholine (0.78 mM in the reaction mixture) in Tris-HCI (0.087 M in the reaction mixture) buffer, pH 7.5. In order to exclude for nonspecific reaction a blank was set for cell lysate, omitting the substrate in the spectrophotomet ric assay. A specific inhibitor, l,5-bis(4-allyldimethylammoniumphenyI)pentane-3-one dibromide, was used to distinguish true AChE activity. Enzyme units are defined as //inn] of substrate utilized per min at 25°C.Protein assay was done according to the method of Lowry et al. (14) with modification (15). Molecular Probes. Molecular probes for c-myc expression (kindly
6327
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20-
provided by Dr. Dalla Pavera) were a 2-kilobase Smal fragment (from pMC41) (16) containing the first two exons of the myc gene and a 1.4kilobase Cla\/EcoRl (pMC41-3RC) fragment containing the third exon of the myc gene. The H4 probe (pHu4A), a gift from Dr. R. G. Roeder, was a 1-kilobase EcoRl/Hintflll fragment cloned in the pBR325 vector (17); N-ras probe (pNPl), kindly provided by Dr. M. Wigler, was a 1kilobase fragment from the first exon of the human N-ras gene (18). Inserts were excised from the vectors with appropriate restriction enzymes (New England Biolabs, Beverly, MA) and electrophoretically purified. Assay for Gene Expression. Dot-blot analysis was performed using total cytoplasmic RNA, prepared according to the method of Favaloro et al. (19) after formaldehyde/standard saline citrate (0.15 M sodium chloride-0.015 trisodium citrate) denaturation (20). RNA was spotted over Gene Screen membranes (New England Nuclear, Boston, MA) using a manifold apparatus (Bio-Rad, Richmond, CA); Northern blot analysis was performed as described by Thomas (21). Molecular hy bridization was carried out at 42°Cfor 24 h in a solution containing 50% formamide, 6x standard saline phosphate EDTA (0.18 M NaCl0.01 M NaPO.,-0.00] M EDTA), 5x Denhardt, denatured herring sperm DNA (200 Mg/ml), total yeast RNA (200 Mg/ml), and labeled probe. Probes were labeled by nick translation with [a-32P]dCTP (3000
ga
ID a x
8-
ag a
4-
"N.
3
Ci/mmol) (New England Nuclear) at a specific activity of at least 3 x 10s cpm/Mg- Blots were washed at a final stringency of 0.1 x SSPE at 60°Cand exposed over Kodak XAR5 films at -70°Cfor 1-2 days with
6-
intensifying screen. Densitometric tracing of dots was performed at 600 nm on a Beckman DU8 spectrophotometer equipped with an automated scanning system. 3o
RESULTS Acetylcholinesterase and Hemoglobin Kinetics of Induction. ara-C treatment of the K-562 cell line can induce the coordinate expression of the erythroid markers AChE and Hb. As shown in Fig. 1, the peak of AChE activity was reached within 4-5 days of induction and remained constant for several days. Hb production followed a similar mode of appearance. During these experiments the cell viability, monitored daily, was always well above 90%. At our level of detection it was impossible to determine if the appearance of one of the two markers was slightly delayed in comparison to the other; our data, however, indicate that the synthesis is virtually synchronous. The ara-C treatment of this cell line was accompanied by deep morphological changes. Fig. 2 shows the K-562 morphol ogy after 5 days of induction (Fig. 2A) as compared to that of untreated cells (Fig. 2B). After benzidine-hematoxylin staining it can be observed that the cells are markedly reduced in size and that the percentage of benzidine-positive cells was increased up to 60%. After 8 days of induction this percentage reached 80%. We then wanted to determine if this type of differentiation was dependent on the cell clone used. Two different K-562 lines, the original K-562 and its subclone K-562 (S), were induced in parallel with our K-562 (S)P subline. The results, summarized in Table 1 indicate that in spite of a different constitutive level, the extent of induction of the two markers was on the same order of magnitude for all three clones. The coordinate expression of AChE and Hb in ara-C-treated K-562 cells was not reversed by drug removal. In fact (Fig. 3), if the inducer was removed at day 4, when the expression of the two markers had reached its plateau, and the cells were shifted to normal medium, both AChE and Hb levels remained constant up to day 12. At this time, since cell viability had lowered to around 82%, the experiment was interrupted. In these experi ments AChE activity and Hb content were measured, respec tively, as units per g of protein and as ng Hb per mg of protein
2468 Days Fig. 1. Kinetics of Hb and AChE induction of K-562 (S)P by ara-C. Hb concentration (A) AChE activity (B), and cell concentration (C) of treated (•) and control cells (A).
in order to account for the cellular debris present in the cell culture after 12 days of induction. The inhibition of the cell renewal capacity of K-562 cells grown in the presence of ara-C is further demonstrated in Fig. 3C, where we assayed the clonogenic activity of cells treated with the drug for different lengths of time. A 24-h treatment was sufficient to inhibit by 76% the ability of the cells to form colonies in semisolid media. After 3 days of induction the cells completely lost this capacity. These experiments suggest that ara-C can exert some kind of "priming" effect over K-562 cells. This is readily demonstrated in Table 2 where we show that cells grown for 24 h in the presence of the inducer and then shifted to normal medium for 6 days could express levels of AChE and Hb as high as those of cells grown for the same time in the continuous presence of the inducer. From these experiments we have concluded that ara-C treat ment can irreversibly activate the differentiative program of K562 cells and that none of the considered parameters, namely cytostatic effect, self-renewal capacity, and erythroid marker expression can be altered by the removal of the drug. DNA, RNA, and Protein Synthesis during ara-C Induction. The major effect of ara-C treatment in terms of DNA, RNA, and protein synthesis was as expected the dramatic decrease of DNA production shortly after the addition of the chemical. In Fig. 4 we show the incorporation of tritium-labeled precursors at various times after induction. The data, expressed as the ratio of incorporated counts of control to that of treated cells, indicate that DNA synthesis was halved after l h of induction
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at various times after induction, was assayed by quantitative dot-blot analysis. In a first round of hybridization we tested probes specific for the 5' or 3' ends of the human c-myc gene
A
(data not shown). The results, shown in Fig. 5/Õ,demonstrate that after only 4 h of induction, the c-myc mRNA synthesis was decreased by about 10-fold as compared to untreated cells. Virtually identical results were obtained using either c-myc probes. Northern blot analysis conducted on selected RNA samples demonstrate that the size of the decreased transcript (2.4 kilobases) was identical to the authentic human c-myc mRNA (Fig. 5A). It should be pointed out that the reduced expression of the c-myc gene occurred independently on the global RNA synthesis (see Fig. 4) since the incorporation of tritium-labeled uridine, in ara-C and control cells, was on the same order of magnitude. Parallel experiments were carried out on total cytoplasmic RNA extracted from K-562 cells whose growth was arrested by serum deprivation ("quiescent" cells). In this case we observed a shallow, progressive decrease of this oncogene expression (Fig. 6). By densitometric tracing of the dots we have estimated that levels of inhibition comparable to those of cells treated with ara-C for 4 h could be obtained after 4 days of starvation. The selective and specific inhibition of c-myc mRNA produc tion during ara-C-induced differentiation was suggested from the experiments summarized in Fig. 7. Total cytoplasmic RNA was hybridized with a human histone 4 probe (Fig. 7A) and with a human N-ras probe (Fig. IB). In both experiments the lack of decrease in the hybridization signal strongly suggests that at the beginning of the cell terminal differentiation the cmyc gene is selectively turned off. As an additional control we have assayed RNA prepared from sodium butyrate-induced K-562 cells. In this case, as was demonstrated for hemin induction (22), no discernible decrease in c-myc synthesis could be observed (Fig. 1C). DISCUSSION
Fig. 2. Photomicrograph of cytocenlrifuged smears of induced (A) and uninduced (B) K-562 (S)P cells. Slides were stained with benzidine-hematoxylin after 5 days of induction, x 1250.
and steadily decreased up to 8 h when it leveled off. To the contrary, both RNA and protein synthesis remained constant during the first 48 h of treatment. Molecular Changes during K-562 Differentiation. Since our data suggest that ara-C treatment of the K-562 cell line is accompanied by the early, irreversible commitment of the cells to terminal differentiation and since almost no changes in the rate of RNA and protein synthesis were detected soon after the beginning of the induction, we considered it an interesting point to search for selective changes in the level of expression of genes related to cell proliferation and/or differentiation. One of the best candidates for this type of analysis is the c-myc oncogene. Total cytoplasmic RNA, extracted from K-562 cells
Although a variety of chemical agents is able to promote hemoglobin synthesis in K-562 cells, only recently has it been demonstrated that irreversible induction of this marker can be obtained by treating this cell line with the cytosine analogue 1/3-D-arabinofuranosylcytosine(8). We demonstrate that the coordinate expression of two erythroid markers (AChE and Hb), obtained by ara-C treatment, is due to a terminal differentiation program. The induction of irreversible differentiation is associated, at the molecular level, with the decreased expression of c-myc, an oncogene involved in normal and neoplastic control of cell proliferation and dif ferentiation (4-7). Other authors reported that, in the murine system, AChE and Hb expression during erythroid cell differentiation follows a sequential pattern (23). Our data, on the contrary, strongly suggest that the expression of these two markers during K-562 differentiation is synchronous. We do not know whether this discrepancy can be attributed, at least in part, to a different mode of erythroid cell commitment in different species or is a characteristic of the K-562 cell line. Although a definitive answer to these questions awaits the isolation of AChE molec ular probes, the almost identical kinetics of the expression of two markers in three different cell subclones can be consistent with the coordinate expression hypothesis. The continuous presence of the inducer was not necessary to achieve terminal differentiation. Drug removal at the time of maximal expression of the markers did not change any of the monitored parameters:
6329
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Table 1 ara-C induction oferythroid differentiation in three K-562 cell clones % of increase
Constitutive value dayS
(units/g protein)3.48 of cloneK-562 Cell ±0.63° (S)P K-562 (S) 1.17 ±0.47 K-562AChE 0.89 ±0.30Hb(¿ig/mg ' Mean ±SD of of at least four determinations.
day 7
of pro tein)5.19 ±1.25 4.06 ±0.90 3.06 ±0.48AChE201
±103 229 ±34 260 ±62Hb216
±47 ±76 118 287+ 118 222 ±97 288 ±55 287 ±126AChE215 374 ±93Hb317± 342 ±55
Table 2 K-562 induction after brief exposure to ara-C AChE activity and Hb concentration were determined after 7 days of incubation timeAChE
Incubation
(units/g protein/cell) AChE control*
h exposure ara-C5.76 to (274)°
h exposure ara-C9.31 to
(320) 2.10(0) 2.91 (0) Hb (pg/ng protein/cell) 30.13(372) 22.4(312) Hb control*24 8.11 (0)168 7.14(0) °Numbers in parentheses, -fold increase x 100 with respect to the control. * The controls did not receive any drug; also, for the 24-h exposure to ara-C, control cells were washed after 24 h and resuspended in fresh medium.
M
5.
•
•v • 4•
e o o
o o
8
24
48
Hours Fig. 4. DNA, RNA, and protein synthesis at various times during ara-C treatment. Results are expressed as a ratio of counts incorporated into control cultures relative to counts incorporated into treated cells. •,DNA; A, RNA; •, proteins.
12 Days Fig. 3. Irreversible induction of K-562 (S)P by ara-C. Cells induced for 4 days were harvested, washed, and resuspended in drug-free medium at the same cell concentration. J, removal of the drug. Hb concentration (A), AChE activity (A), cloning efficiency (C), and cell concentration (D) of treated (•)and control cells (A).
ara-C-induced differentiation of K-562 cells differs from in vivo differentiation since it is accompanied by mitotic arrest, whereas in vivo and also in the MEL cell system erythroid differentiation is accompanied by limited but active cell prolif eration. Thus ara-C can initiate an irreversible cascade of events leading to the reduction of the pluripotent properties of this cell line in sharp contrast to what is observed after hemin or sodium butyrate induction. The nature of the signal that con trols the sequential and/or coordinate activation of genes re sponsible for erythroid differentiation is still an unresolved problem. Such an "inducer" could act by activating one or
several factors that are responsible for the transcription of the genes coding for the erythroid phenotype. In a recent study, by genetically manipulating the regulation of differentiation in MEL cells, convincing evidence is provided that transacting cytostatic effect; self-renewal capacity; and erythroid markers factors are involved in the regulation of globin and non-globin expression. Moreover, the peculiar mode of differentiation in genes by interaction with sequences responsible for creating duced by ara-C in K-562 cells is even more evident if we consider DNase I-hypersensitive sites surrounding these genes (24-26). that a 24-h treatment with the drug is sufficient for the irrever It would be interesting to investigate whether ara-C mimics one sible activation of the differentiation program. of the factors necessary for triggering erythroid differentiation 6330
K-562
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c-myc EXPRESSION
HOURS
0
12
8
24
48
12
18
24
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72
96
72
Fig. 6. c-m>>cmRNA levels of "quiescent" cells. RNA was prepared in parallel with that of induced cells. Experimental conditions were identical to those of Fig. 5/4.
1
B
HOURS 24
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I
*
25 HOURS
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5 2.5 Fig. 5. A, c-myc mRNA levels during ara-C induction assayed by dot-blot hybridization. For each dot, 5 to 0.5 fig of RNA were applied to Gene-Screen membranes. B, Northern blot analysis of selected RNA samples prepared from ara-C-induced K-562 cells. Each lane contains 20 ¿igof total cytoplasmic RNA; agarose concentration was 1.5%. The endogenous rRNA was used as internal standard for size and equal sample concentration. The probe used was pMC4l3RC containing only the third exon. KB, kilobase.
or if its mode of action is an accessory feature of its pharma cological properties. Additional experiments will be needed to answer this question. Terminal differentiation induced by ara-C is characterized by a peculiar change at the molecular level: the marked decrease of c-myc mRNA. The down regulation of this oncogene, in association with the appearance of the differentiated phenotype, has been described in several other biological systems (4-6), whereas its increased expression has been found to be correlated with the proliferative status of many cell types (7). In the K562 cell line the c-myc gene is constitutively expressed at levels comparable to several other neoplastic cell lines according to Westin et al. (5) and to our findings.4 The sharp decrease in the
12 0.6 Fig. 7. Histone 4 (A) and N-ras (A) mRNA levels after ara-C induction; cmyc (C) expression in K-562 cells induced with sodium butyrate. Experimental conditions were identical to those of Fig. 5A.
irreversible erythroid differentiation of the K-562 cells and with the irreversible arrest of cell proliferation, c-myc expression was dramatically reduced after the first 4 hours of induction with ara-C; this finding is a further confirmation that this oncogene plays a key role in the early stages of differentiation, well before the final cell commitment has occurred. We did not observe a statistically reproducible biphasic change in c-myc production, as described for MEL cells (4); decreased expres sion of c-myc was comparable to that described in other cellular systems (5, 27, 28). If c-myc regulation was a cell cycle-related event induced by the cytostatic properties of ara-C, we would expect parallel changes in the H4 mRNA synthesis. The expression of this expression of this oncogene correlates with the induction or histone gene was unchanged during the first 4 hours of ara-C 4 Unpublished observations. treatment, when DNA synthesis is still active, strongly suggest6331
K-562 TERMINAL
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ing that c-myc control of transcription, in our cell clones, was dependent on the activation of a differentiative program. The reduced expression of the c-myc gene was not just a consequence of a general reduction in total RNA production since the level of incorporation of labeled precursors and the expression of another oncogene, N-ras, were not altered during the first 48 h of ara-C treatment. Hemin, a chemical known to induce erythroid differentiation of K-562 cells, did not alter c-myc expres sion, as already reported by other investigators (22); also, sodium butyrate, which has broader but still reversible differ entiative properties, was unable to inhibit c-myc expression. This result further suggests that only factors able to interact with genes active in the early moments of cell differentiation will eventually trigger the cascade of events leading to the expression of a specialized cell function. ACKNOWLEDGMENTS We are deeply grateful to all the colleagues who have provided molecular probes and cell lines. We are particularly grateful to Prof. S. Ottolenghi for suggestions and criticisms. Thanks are given to Aldo Profumo and to Paola Origone for expert technical assistance.
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