the c-myc oncogene is regulated independently of differentiation in myeloid cell lines

Leukemia Research Vol. 13, No. 8, pp. 651-659, 1989. 0145-2126/89 $3.00 + .00 Printed in Great Britain. Pergamon Press plc

T H E c-myc O N C O G E N E IS R E G U L A T E D I N D E P E N D E N T L Y OF D I F F E R E N T I A T I O N IN M Y E L O I D CELL LINES*

PAMELA ROBERTS, MARK JONES, ROSEMARY GALE, SHAUN THOMAS, NICHOLAS TIDMAN and DAVID LINCH

Department of Haematology, University College, London, U.K.

(Received 7 October 1988. Revision accepted 8 March 1989)

Abstract--Human myeloid leukaemia (U-937 and HL-60) cells when incubated at low cell densities with human recombinant ),-interferon underwent functional maturation without any loss of proliferative potential relative to uninduced cells. In addition, the proportion of cells in S,G2/M and levels of c-myc oncogene (mRNA and protein) were maintained at the same level as those of untreated control cells. However, cells grown under similar conditions but with retinoic acid matured to the same extent but became growth inhibited with concomitant reductions in the proportion of cells in S,G2/M and levels of c-myc mRNA and protein.

These studies indicate firstly that c-myc levels are regulated independently from differentiation in myeloid (non lymphoid) cells, secondly that ),-interferon can induce differentiation without growth arrest under conditions of low cell density and thirdly emphasise the close association of c-myc expression with proliferative capacity.

Key words: c-myc, gamma-interferon, HL-60 cells, U-937 cells, differentiation, cell-cycle, proliferation.

I N T R O D U C T I O N

THE TWIN processes of myeloid proliferation and differentiation are intimately linked in vivo. The proliferation of primitive myeloid cells is stimulated at least in part by a series of growth factors (colony stimulating factors) and the process is associated with progressive maturat ion of each generation of daugh- ter cells. In the neutrophil series cell division ceases at the myelocyte stage [1] and only then does further differentiation occur without proliferation. In the monocyte series, some proliferation may take place in tissue macrophages [2]. The mechanisms of proliferation and differentiation are difficult to analyse using fresh bone marrow cells because of the marked

* This work was supported in part by grants from the University College Hospital Trust and the Kay Kendall Leukaemia Fund.

Abbreviations: c-myc, c-myc oncogene; DNA, deoxy- ribonucleic acid; ),-IF, recombinant gamma interferon; m- RNA, messenger RNA; NBT, nitro blue tetrazolium; PBS, phosphate buffered saline; RA, retinoic acid; RPMI medium, Rothwell Park Memorial Institute medium; S, G2/ M, synthesis, post-synthesis, pre-mitosis/mitosis, phases of the cycle; SSC, SSPE, salt solutions; SDS, sodium dodecyl sulphate; TPA, 12-O-tetradecanoylphorbol 13- acetate.

Correspondence to: Dr P. J. Roberts, Dept of Clinical Haematology, UCMSM, 98 Chenies Mews, London WC1E 6HX, U.K.

651

heterogeneity of this cell population. For this reason, many studies have utilised myeloid leukaemic cell lines and in particular the HL-60 human promyelo- cyte leukaemic cell line [3]. A wide range of chemicals and pharmacological agents can induce differentiation in these cells, including dimethyl sulph- oxide, phorbol esters, retinoic acid, cyclic adenosine monophosphate and dihydroxyvitamin D 3 [4].

Differentiation with these agents is associated with arrest of cell growth. This growth arrest could be due to the fact that the uninduced HL-60 cell is a relatively late cell in the differentiation pathway and further maturation quickly takes the cell to the stage at which its normal counterpart ceases to proliferate. Alternatively, the growth arrest might represent simple toxicity of the inducing agent, with differentiation merely representing a response to sub- lethal damage.

Proliferating cells express high levels of c-myc oncogene m R N A and this is particularly so in HL- 60 cells in which there is amplification of the c-myc gene [5-7]. The c-myc oncogene has been implicated in the process of D N A replication [8, 9] but experiments with fibroblasts indicate that high levels of c- myc expression are not a prerequisite for mitogenesis [10]. It has also been suggested that high levels of c- myc prevent differentiation and that down regulation of c-myc is required to enable maturation to occur.

652 PAMELA ROBERTS et al.

This is supported by the finding that in HL-60 cells the differentiating agents discussed above lead to down regulation of c-myc expression as well as inhibition of cell growth [7, 11-18]. In addition, the constitutive expression of c-myc m R N A in murine erythroleukaemia cells [19] and v-myc m R N A in U- 937 cells blocks terminal differentiation [20].

Gamma interferon (),-IF) is a lymphokine which is available in recombinant form for laboratory and clinical use. It has been shown to induce differentiation in both HL-60 [21] cells and in cells of the primitive monocyte line U-937 [22-24]. The reported effects of ),-IF on cell growth are con- troversial (see [25] for summary). Several authors have demonstrated differentiation of HL-60 cells accompanied by inhibition of cell growth in response to ),-IF [26-28]. Similar effects were reported in U- 937 cells [24, 27]. Kronke et al. [29] found, by contrast, that ),-IF did not inhibit growth of HL-60 cells and did not down regulate c-myc R N A expression although evidence of differentiation was not provided. Others have shown that differentiation of HL-60 cells in response to ),-IF begins within 24 h of ),-IF exposure and that c-myc m R N A expression is not reduced at this time [30-31]. Growth inhibition and down regulation of c-myc expression occurred at 72 h [32]. Similarly Dayton et al. [33] found that the proliferative ability of HL-60 cells was not reduced until after 3-day exposure to ),-IF.

In the present study we demonstrate that U-937 cells as well as HL-60 cells, (when grown at low cell concentrations), can be induced to differentiate by recombinant ),-IF and this occurs without growth inhibition or detectable down regulation of c-myc m R N A or c-myc protein content over a period of five days. Down regulation of c-myc is not therefore, a prerequisite for myeloid differentiation.

M A T E R I A L S A N D M E T H O D S

Cell lines Stock cultures of the human promyelocytic HL-60 [3] and

monocytoid U-937 [22] leukaemia lines were maintained at a cell density of between 1 and 5 x 105 cells/ml in RPMI medium supplemented with 10% foetal calf serum in a fully humidified atmosphere of 5% CO2.

Differentiation induction Differentiation was induced by resuspending cells (1-

7 x 104/ml) in fresh medium containing either recombinant gamma interferon (200 U/ml, Biogen Research, Cambridge, MA), or retinoic acid (1 ~M, Sigma Chemical Co., Poole, Dorset). Cells were grown for 4-6 days without replenishing the medium.

Functional maturation Cell maturation was assessed by an adaptation of the

nitroblue tetrazolium (NBT) test [34]. Cells were incubated (37°C, 30 min) in 0.05% (w/v) NBT in RPMI medium containing 12-O-tetradecanoylphorbol 13-acetate (TPA) 1 ~g/ml to stimulate the respiratory burst. Control samples were incubated without TPA to measure the resting level of NBT reduction. Positive cells containing black deposits of formazan were identified by light microscopy. The percentage of positive cells was calculated from a total of 300 cells in each sample. Cell viability was assessed by trypan blue exclusion.

Estimation of c-myc protein content (a) Flow cytometry. Cell samples (1-2 x 106 cells) were

fixed in formaldehyde in phosphate buffered saline (PBS) and then permeabilised in absolute methanol at 4°C for 30 min. After washing in 0.1 M Tris buffer pH 6, the cells were incubated for 20 min at 37°C in Tris buffer pH 6 containing 0.1% RNase (Type IIIA Sigma). The cells were then washed in Tris buffer and the cells used for either c- myc protein estimation or cell cycle analysis.

The c-myc protein content was determined by incubating the cells for 60 min on ice with the monoclonal antibody DCM 905 (obtainable as monoclonal antibody CT14, Cam- bridge Research Biochemicals Ltd) at a concentration of 20 ~tg/ml. The cells were then washed three times and incubated for 45 rain on ice with FITC-rabbit anti-mouse immunoglobulin (Dako Patt Ltd). After three washes the mean cell fluorescence was determined on a FACS IV (Becton-Dickinson, Mountainview, California) by cal- culating:

Mean cell fluorescence

channel number x channel contents =E total cells

Specific staining was determined by subtraction of the mean cell fluorescence obtained using an irrelevant murine" monoclonal antibody (CD3) as the first layer. The DCM 905 monoclonal antibody was produced against a synthetic peptide with the sequence of the C-terminal amino acids 408-439 of p62 cmyc [35]. Fluorescent microscopy revealed that c-myc staining of HL-60 and U-937 cells was almost entirely nuclear, and could be blocked by addition of the specific c-myc peptide.

(b) Western blotting. U-937 cells were pelleted and lysed by resuspending directly in SDS gel sample buffer. Protein from 2 x 105 cells was loaded onto each lane of a 10% polyacrylamide SDS gel containing 38:1 acrylamide:bis- acrylamide [36]. Proteins were then transferred onto nitrocellulose (HybondC Extra, Amersham International) by semi-dry blotting (Atto, Japan) at 0.5 A for 45 min in 25mM Tris, 197mM glycine, 20% methanol. After blotting, the filter was immersed in blocking solution (10% non-fat dried milk (Marvel), phosphate buffered saline (PBS)) and agitated for at least 1 h at room temperature. Duplicate blots were then incubated overnight at 4°C with either a mixture of anti c-myc mouse monoclonal anti- bodies, 1:100 dilution CT14, 6El0, 9E10 (Cambridge Research Biochemicals Ltd, England); or with anti-tubulin antibody, 0442 anti-peptide antibody from rabbit (Dr P. Parker, Ludwig Institute, London) (1 : 100 dilution in 10% non-fat dried milk, PBS, 0.05% Tween-20 (Sigma)). After washing with three changes of PBS, 0.05% Tween-20, the filters were incubated for 1 h at room temperature with a second [125I] sheep-anti-mouse antibody (16~tCi/~tg, Amersham International) or [125I] donkey-anti-rabbit anti-

c-myc Expression and myeloid cell differentiation 653

body (20 ~tCi/~tg, Amersham International), respectively at 0.2 ttCi/ml in 10% Marvel, PBS, 0.05% Tween-20. The filters were again washed with three changes of PBS, 0.05% Tween-20, dried and exposed to pre-flashed Hyperfilm-MP X-Ray film (Amersham International). Rainbow markers (Amersham International) were used as protein standards.

Measurement o f c-myc rnRNA Total RNA was prepared from 107 cells using a single-

step guanidinium thiocyanate method [37]. Twenty-micro- gram aliquots were glyoxylated and electrophoresed in a 1.4% agarose gel in 10 mM phosphate [38]. The RNA was then transferred onto a nylon filter (Biodyne, Pall Corp.) by capillary blotting in 20 x SSC [39]. The filter was pre- hybridised for 4 h at 42°C in 25% formamide, 5 x SSPE, 5 x Denhardts, 0.2% SDS, 2001xg/ml single-stranded salmon sperm DNA and then hybridised overnight at 42°C with 32p oligo-labelled probes concurrently for c-myc (exon 3) and y-l.1 (a gamma-interferon-inducible gene from Dr B Bloom, Albert Einstein, New York) [40] and then for actin, the latter to check the equivalence of RNA loading. Hybridisation was in 50% formamide, 5 × SSPE, 1 x Denhardts, 0.1% SDS, 200~tg/ml single-stranded salmon sperm DNA. After washing at 55°C in 1 x SSC, 0.1% SDS then 0.1 x SSC, 0.1% SDS the blot was exposed to X-ray film (Hyperfilm-MP Amersham International) overnight at -85°C. All solutions were prepared as described in Maniatis et al. [39].

Cell cycle analysis Fixed, permeabilised and RNase treated cells were resus-

pended in 400 Ixl of PBS containing 0.05 mg/ml of pro- pidium iodide and incubated for 10min at room temperature prior to analysis on a FACS IV.

R E S U L T S

HL-60 and U-937 cells seeded at low cell concentrations (mean cell density 4 × 104 cells/ml) pro- liferated in culture over 5 days with growth increments of 20-fold and 30-fold, respectively. Growth increments were inversely proport ional to the seeding density as shown in Fig. 1 for U-937 cells. Replicate cultures of HL-60 and U-937 cells were grown under identical conditions but with the addition of either y-interferon (200 U /ml ) or retinoic acid (1 ~tM), to induce differentiation. Analysis of the U-937 cultures after 5 days growth in the presence of y-interferon showed no significant inhibition of growth compared to control cells (Table 1); however, cells induced with retinoic acid were growth inhibited without any reduction in cell viability, and there was a concomitant reduction in the percentage of cells in the S and G2/M phase of the cell cycle. In an experiment where measurements were made daily, growth inhibition of retinoic acid-induced cells was apparent by day 3 (Fig. 2a) and there was also a reduction in the percentage of cells in S ,Gz /M compared to control cultures at this t ime (Fig. 2b) whereas both cell number and the percentage of cells in cycle were

6O

C

4o

20

Initial cell density (x 104/ml)

FIG. 1. The proliferation of U-937 cells was dependent on the initial cell density. Growth increment is defined as the cell concentration after five days growth, divided by the

initial cell density.

TABLE 1. GROWTH AND CELL CYCLE ANALYSIS OF U-937 CELLS AFTER 5 DAYS IN CULTURE

Treatment Cell number Cells in S,Gz/M (% of uninduced control)

Control 100 100 ),-IF

(200 U/ml) 92 + 5* 93 - 5 RA

(1 ~tM) 44 ± 6 63 ± 4 No. of experiments 7 3

* The data given are the mean -+ 1 S.E. The absolute number of cells in the control cultures was

8x105 /ml+- I ( m e a n ± l S.E.) and of these 3 3 % ± 4 were in S,G2/M.

maintained at control levels in y-IF-induced cultures. In experiments with HL-60 cells, the cell concentration after induction for 5 days with y-interferon was 123 _+ 15% of control (mean _+ 1 S.E. , n = 3) whereas the concentrat ion in RA-induced cells was 45 _+ 8% of control. Despi te their differential effects on cell growth, both gamma interferon and retinoic acid induced the same degree of functional maturation of both cell lines, as determined by the presence of TPA-st imulated reduction of nitroblue- tetrazolium, an indicator of phagocyte oxidase activity (Table 2).

c-myc Protein

Levels of c-myc protein in U-937 and HL-60 cells were est imated by immunofluorescent staining with anti c-myc antibody. There was little down regulation


g ~ A

(9

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t~L~

o.=

- - v -$ L~

0

40

a)

I I I 2 4 6

2O

10 b)

i i i

2 4 6

Incubation time (days)

FIG. 2. (a) Proliferation of U-937 cells and (b) proportion of cells in S and G2/M stage of the cell cycle during differentiation induced by either y-interferon (O) (200 U/ ml) or retinoic acid (&) (1 ~tM), compared to untreated control cultures (0) . Data are from a single experiment.

TABLE 2. F U N C T I O N A L M A T U R A T I O N OF HL-60 AND U -

937 CELLS I N D U C E D BY y - I N T E R F E R O N AND RETINOIC ACID

M E A S U R E D BY T H E T P A - S T I M L r L A T E D R E D U C T I O N OF NBT

Inducer HL-60 U-937 % NBT positive cells

Control 14 +- 9* 10 + 4 y-Interferon (200 U/ml) 57 + 13 48 -+ 5 Retinoic acid (1 ~tM) 43 + 10 53 + 7

* The data given are the mean + 1 S.E. of three experiments with HL-60 cells and seven experiments with U-937 cells•

TABLE 3. S E M I - Q U A N T I T A T I V E ESTIMATION OF c-myc PRO-

TEIN CONTENT

Inducer HL-60 U-937 (% of uninduced control)

y-Interferon 130 -+ 37 158 +-- 85 Retinoic acid 16 -+ 6 18 -+ 4

The data given are the mean -+ 1 S.E. of three experiments. Cells were induced to differentiate for 5 days with either y-IF (200 U/ml) or retinoic acid (1 ~tM). The specific anti-c-myc staining of U-937 cells was less (56 -+- 36%) than that of HL-60 cells.

125

~ 100

o O 7 5 ~ k

E d "6 so

g

I I = i 1 2 3 4

=_=

o

E.6

175

150

125

100

50

25

I J I I I

1 2 3 4 5

Incubation time (days)

FIG. 3. c-myc protein levels measured by immuno- fluorescence, in untreated control cultures of HL-60 and U-937 cells (0 ) or cultures induced to differentiate with either y-Interferon (O) (200 U/ml) or retinoic acid (lit),-

(1 ~tM). Data are from a single experiment.

of c-myc prote in in cultures that were induced to differentiate with y- interferon c o m p a r e d to uninduced control cultures, whereas levels of c-myc protein were marked ly reduced in cells that were grown with ret inoic acid (Table 3). A daily analysis of c-myc levels during different iat ion showed little change over the first two days in cul ture with ret inoic acid fol lowed by a sharp decrease in c-rnyc prote in by

day 4 (Fig. 3). There was some fluctuation in the expression of c-myc prote in in y- in ter feron- t rea ted cells but no significant decrease in levels with t ime were detected. The ma in tenance of c-myc protein levels in y- IF- induced cells and down-regula t ion in retinoic acid induced cells was conf i rmed by western blotting (Fig. 4). I t should be no ted that the anti c-myc ant ibody reacts with several minor bands of

® 1 2 3

m w

6 7 -

4 3 -

N

3 0 -

FIG. 4. (a) c-myc Protein levels (indicated by arrow) in U- 937 cells measured by western blotting, (b) tubulin levels detected in the same electrophoresis gel. Lane (1) retinoic acid-induced, lane (2) untreated, lane (3) y-interferon-

induced U-937 cells.

655

® H L - 6 0

1 2 3

u - g 3 7

1 2 3

1-6 k b -

1.1 k b -

®

1.6 k b -

FIG. 5. (a) c-myc mRNA Measured by northern blotting in HL-60 and U-937 cells cultured for five days. Lane (1) untreated cells, lane (2) cells cultured with retinoic acid (1 txM), lane (3) cells cultured with y-interferon (200 U/ ml). The induction of the 1.1kb y-interferon-inducible gene [40] to levels above control, demonstrated that y- interferon was active in these experiments. (b) Actin mRNA in HL-60 and U-937 cells measured by northern

blotting. The order of the lanes is as shown in (a).

656


unknown nature in addition to the major p62 band. This is in contrast to Daudi cells (B cells) in which a single band is observed (data not shown).

c-myc m R N A

Steady state levels of c-myc m R N A were measured in RNA from cells induced for 5 days with either RA or y-IF by northern blotting (Fig. 5). The amount of c-myc mR NA in cells induced with y-IF was similar to that in control cells but was much reduced in cells that had been cultured with retinoic acid, results which correlate with those observed for c-myc protein. Functional analysis of the cells used for these experiments confirmed that maturat ion had occurred in both y-IF and RA-induced cells (49% of retinoic acid-induced cells and 41% of y-IF-induced cells were positive in the NB T test, compared to 2% of control cells). Actin m R N A probed as a standard to check for total R N A loaded on the gel confirmed that the amounts were approximately equal.

DISCUSSION

The levels of c-myc protein and c-myc m RN A did not decline in y-IF induced U-937 and HL-60 cells, which is in contrast to the retinoic acid induced cells which showed a similar degree of differentiation but were partially growth arrested. Regulation of c-myc mRNA in HL-60 cells is predominantly at a transcriptional level [17, 18] and retinoic acid has been shown to cause a block in the elongation of the c- rnyc RN A transcript between exons 1 and 2 [14]. The finding that functional maturation can occur in myeloid cells without down regulation of c-myc is apparently at variance with the finding that constitutive expression of v-myc in U-937 cells appears to reverse phorbol-induced maturation [20]. This situa- tion is not directly comparable to our experiments as the levels of myc m R N A produced in the transfected cells is grossly in excess of the usual levels found in the undifferentiated non transfected cells. Our findings support the concept that there is a close association between c-myc expression and the rate of cell proliferation and indicate that down regulation of c-myc is not a prerequisite for myeloid differentiation.

These studies show that y-IF can induce functional maturation of both U-937 and HL-60 myeloid cell lines and that this can occur over a five day period without any decline in the proliferative status of the cells. The y-IF-induced inhibition of HL-60 and U- 937 cell growth reported by others [24, 26-28] is probably related to the high cell concentrations used in those studies. We show here that the proliferation of U-937 and HL-60 cells is highly sensitive to the seeding density used with relative inhibition of cell growth at higher cell concentrations. This could be due to consumption of vital nutrients or to production of inhibitory metabolites. It is well recognised that y-IF induces myeloid cells to produce biologically active compounds such as tumour necrosis factor [41] and it is likely that the previously reported effects of y-IF on HL-60 cell growth represent such indirect effects.There have been far fewer data reported on the effects of y-IF on U-937 cell growth and differentiation but this is a valuable myeloid model in that there is no amplification of the c-myc gene.

The finding that y-interferon can induce differentiation without inhibition of myeloid cell growth may have implications for the clinical exploitation of y-IF in the t reatment of certain phagocyte defi- ciencies such as chronic granulomatous disease [42]. The induction of differentiation without growth arrest by y-interferon in two myeloid leukaemic cell lines also provides an excellent model for dissecting the biochemical events associated with the differentiation and maturat ion process.

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