Induction of differentiation in HL60 leukaemic cells: A cell cycle dependent all-or-none event

Leukemia Re.seur('h Vol. 8. No. I, pp. 27--43, 1984. Printed in (ireal Hrilain.

0145-2126/8453.0(1 + 0.(N) it:) 1984 Pergamon P r e ~ Lid.

INDUCTION OF DIFFERENTIATION IN HL60 LEUKAEMIC CELLS: A CELL CYCLE DEPENDENT ALL-OR-NONE EVENT* ANDREW W. BOYD and DONALD METCALF Cancer Research Unit, Walter and Eliza Hall Institute, Post Office Royal Melbourne Hospital, Victoria 3050, Australia (Received 8 July 1983. Accepted 19 July 1983)

Abstract--The human leukaemia cell line (HL60) shows a limited capacity to differentiate spontaneously, but this property can be greatly enhanced by chemical inducers. Sodium butyrate induced differentiation in virtually 100ale of HL60 cells over a four-day interval to cells with multiple phenotypic markers of monocytes. Clonogenic analysis in agar demonstrated that differentiated cells (either spontaneous or induced) irreversibly lost clonogenic potential. This appeared to be an all-or-none process with unaffected cells exhibiting unaltered clonogeneity. A study of the kinetics of colony formation showed that most, if not all, cells completed one division in the presence of butyrate and sometimes several divisions before loss of proliferative potential. Despite the uniform spectrum of cell cycle states present in HL60 cultures when butyrate was added, all differentiated cells were shown to' be arrested in GI. Evidence was obtained suggesting that the 'switch' into the differentiation pathway occurred during a restricted stage of the cell cycle, either late in the cycle (G2-M) or early in GI. Key words: Leukemia, induced differentiation, butyrate, HL60, clonogenicity.

INTRODUCTION THE MOLECULAR mechanisms which control cell differentiation and proliferation are still largely u n k n o w n . In some cases a definite stimulus is required to initiate proliferation (e.g. antigen or mitogen binding to l y m p h o i d cells). H o w e v e r , even in these situations the chain o f events which link the event o f binding o f a ligand to its receptor with an alteration in the nucleus resulting in either initiation o f cell division or differentiation remain poorly u n d e r s t o o d [9, 26]. One family o f molecules which might control these events is the cellular protein kinases, and in two situations, epidermal g r o w t h factor [14] and nerve g r o w t h factor [20], the rate o f proliferation has been s h o w n to alter in response to application o f the specific h o r m o n e to an a p p r o p r i a t e target cell. H o w e v e r , n o conclusive evidence is yet available to show that protein kinases are the critical ' s e c o n d messengers' and not simply changes which parallel events involved in signal transmission. A n o t h e r a p p r o a c h to the study o f control o f proliferation and differentiation would be to bypass these intervening cytoplasmic processes and induce alterations in the cell nucleus which directly influence proliferation and differentiation. Such experiments might reveal how regulation o f proliferation is mediated in its final stages, and hence c o m p l e m e n t the studies o f m e m b r a n e and cytoplasmic processes. One apparently obligatory m a r k e r o f 'active' areas o f the g e n o m e is the presence o f D N A s e I hypersensitive sites [10]. D N A h y p o m e t h y l a t i o n is correlated with b o t h gene activation and D N A s e I sensitivity [11, 18, 19, 24, 27, 35] D e m e t h y l a t i o n o f previously methylated D N A can be induced by exposure *This work was supported by the Lyndal Skea Leukaemia Research Fund, The Anti-Cancer Council of Victoria, Australia, The National Health and Medical Research Council, Australia and by Grant No. CA25972 of the National Cancer Institutes, Bethesda, U.S.A. Abbreviations: DMSO, dimethylsulphoxide; FA CS, fluorescence-activated cell sorter; FCS, foetal calf serum; MGG, May-Grtinwald-Giemsa; SRBC, sheep red blood cell; TPA, 12-O-tetradecanoyl-phorbol-13-acetate. Correspondence to: Dr. A. Boyd, Cancer Research Unit, Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia. 27

28

ANDREWW. ~YD and DONALDMETCALF

of the cell to the drug 5-azacytidine and this leads to the expression of hitherto silent genetic information and in some cases, induces changes consistent with differentiation in the treated cells [5, 21]. These newly demethylated regions of D N A have been shown to be susceptible to DNAse I [27]. A further correlation with gene activation is hyperacetylation o f histone proteins [8]. Furthermore, as might be anticipated, DNAse I has been shown to release preferentially a hyperacetylated fraction o f the histone proteins from chromatin [36]. In this context, sodium butyrate induces rapid hyperacetylation o f chromatin [29] and, while other effects of sodium butyrate are observed, the only consistent change seen which might mediate this effect is inhibition o f histone deacetylase [3, 6]. A feature o f butyrate and other chemical inducer mediated effects is that they appear to be cell lineage-specific [2, 23, 25, 31] Thus it appears that butyrate can bypass some o f the intervening steps required by physiological regulators and induce 'appropriate' differentiation in cell lines. We have attempted to explore the process o f differentiation in the HL60 promyelocytic leukaemia cell line. This cell line is known to differentiate in response to several chemical stimuli [12, 17, 30, 34] and indeed one report [12] had documented the capacity o f sodium butyrate to induce HL60 differentiation. Thus HL60 taken together with the wellcharacterized metabolic action o f sodium butyrate and the rapid reversal o f its effects after washing, allowing studies o f any irreversible long-term effects induced by butyrate, made this an ideal combination in exploring this approach to the study o f haemopoietic cell differentiation. In this report we show that exposure of HL60 cells to sodium butyrate results in differentiation into cells which closely resemble normal monocytes. Also, as is the case with the normal g r a n u l o c y t e / m a c r o p h a g e lineage, this was paralleled by irreversible loss in the differentiated cells o f the ability to proliferate. These changes also appeared to be characteristic o f spontaneously differentiated cells normally found in small numbers in HL60 cultures. We also show that the ability of butyrate to induce differentiation in HL60 cells appears to be limited to one part of the cell cycle and, that its mechanism of action may be to increase the probability o f an already existing capacity to differentiate spontaneously into mature myeloid and monocytoid cells.

MATERIALS AND METHODS HL60 cell line

The HL60 promyelocyticleukemia was obtained from Dr R. C. Galio (National Cancer Institutes, Bcthesda, Maryland). The cells were recloned in agar and one of these clones was used in the studies described in this report. The cells were grown in liquid culture in RPMI 1640medium supplemented with 10070selected foetal calf serum (FCS). Agar cultured cells were grown in medium consisting of one part double strength modified Dulbecco's Modified Eagles Medium supplemented with 40070FCS and one part 0.6070agar in distilled water. In this medium control HL60 cells routinely produced colony numbers equal to 50-80070of the input cell number. Colony numbers were linearly related to input cell number over the range 10-1000cells/ml agar culture (this linearity at low cell numbers was highly dependent on FCS batch). Chemical inducers

The chemicals used were obtained from Sigma Chemical Company, St. Louis, Missouri, U.S.A. Large batches of each inducer were prepared to minimize variation in preparations. Sodium butyrate was prepared from butyric acid and 100 mM stock solutions obtained by dilution in Eisen's balanced salt solution. Retinoic acid was dissolved in ethanol at concentrations which ensured that the volume of the stock retinoic acid solution added represented less than 1/1000th of the final culture volume. Both these reagents were aliquoted and stored at -80°C. 12-O-tetradecanoyl-phorbol-13-acetate(TPA) was dissolved in dimethyl sulphoxide (DMSO) at 1 mg/ml and the stock solution stored at -80°C. Dilutions in normal saline were prepared immediately before each experiment. Staining techniques (a) May-Granwald-Giemsa (MGG). Cytocentrifugesmears were routinely prepared from all ceil suspensions

and then stained with May-Gr0nwald-Giemsa. (b) Non-specific esterase stain. The a-napthyl butyrate stain was employed and was performed by Ms. Sue O'Mahoney of the Haematology Department, Royal Melbourne Hospital, Australia.

Differentiation induction by sodium butyrate in HL60 cells

29

(c) Hoechst 33342 stain. Solutions were freshly prepared before each experiment. Cell suspensions at 5 x 10~/ml were incubated with the stain (at 2 )ag/ml) for 90 min in DME supplemented with 2% FCS at 37°C. The cell suspensions were washed and held at 4°C in a container sealed against the light.

A n tisera Monoclonal antibodies. OKMI and OKT9 antibodies were obtained from Ortho Diagnostics, Australia. OKMI is principally reactive with monocytes and macrophages and weakly positive on granulocytes. OKT9 is specific for the transferrin receptor which is a marker of proliferation. FMC 10, 13, 17 and 34 antibodies were the gift of Dr H. Zola, Flinders Medical Centre, Adelaide, Australia. FMC 10, 13 and 34 were principally reactive with polymorphs. FMC 17 was shown to be reactive with adherent macrophages but weakly or unreactive with non-adherent macrophages and monocytes (G. F. Burns, personal communication). Rabbit anti-mouse antiserum A rabbit anti-F(ab ')2 fragment of mouse IgG antiserum was prepared by immunizing rabbits with 100 Bgm of F(ab ')2 fragments of mouse lgG (prepared by pepsin digestion of the Staphylococcal Protein A-Sepharose 4B binding fraction of mouse serum) mixed with Complete Freund's Adjuvant. The antiserum obtained after three immunizations at weekly intervals had reactivity to both mouse x- and k- light chains and the titre was relatively unaffected by the isotype of the test mouse immunoglobulin as assessed by gel precipitation. Specific antibody against mouse F(ab ')~ fragments was purified by absorption to a column consisting of mouse lg-Sepharose 4B and subsequent elution with 0.2 M glycine HCI (pH 2.3). F(ab ')~ fragments of this material were prepared by pepsin digestion and this material was conjugated with fluorescem [15]. Fluorescent antibody ,staining Cell suspensions were washed twice and incubated with mouse monoclonal antibody on ice for 60-90 min at 4 ° C. In the case of OKM 1 and OKT9, 1 Bg of purified antibody was used per million cells. The FMC monoclonal antibodies were added in the form of culture supernatants, and usually 50--100 BI gave maximal fluorescence. The unbound monoclonal antibody was removed by washing the cells and centrifuging them through an FCS underlayer. The cell pellet was resuspended in medium containing a 1:50 dilution of fluorescein-conjugated rabbit anti-mouse Ig antibody (this was approx, five-fold greater than the lowest dilution giving maximal binding). The cells were held on ice for a further 30 rain, washed and resuspended in human tonicity phosphatebuffered saline (HTPBS) supplemented with 5% FCS and 10 mM sodium azide. ,4nal.vsis and sorting of fluorescence-labelled cells Analysis and sorting of cells labelled with fluorescein-conjugated antibody was performed on the FACS 11 (Becton-Dickinson) fluorescence-activated cell sorter. To check that the distribution of antibody binding was restricted to the membrane the cells were also examined by fluorescence microscopy. Phagocytosis by HL60 cell suspensions Two methods were used to test for phagocytic function. (a) Bacterial phagocytosis. Cowan I strain Staphyioccus aureus (10 BI of a 10% v/v suspension) were mixed with 2 x 10-~HL60 cells. The mixture was held at 37"C for 30 rain. The cells were washed twice and centrifuged through underlayers of FCS. Cytocentrifuge smears were prepared and stained with MGG. The cells were examined for ingestion of bacteria. (c) Phagocytosis of opsinized sheep red ceils. This was performed as described previously [5]. In brief, sheep red blood cells (SRBC) pre-coated with rabbit anti-SRBC antibodies were mixed with HL60 cells. After 1 h the unphagocytosed red cells were lysed in hypotonic (0.83*/0 ammonium chloride) medium. The HL60 cells were washed, cytocentrifuge smears prepared and stained with MGG. SRBC which had been phagocytosed could then be identified in the cytoplasm.

RESULTS

Treatment o f H L 6 0 cells with s o d i u m b u t y r a t e induces m o n o c y t i c changes A s o u t l i n e d a b o v e s o d i u m b u t y r a t e h a s p o t e n t i a l a d v a n t a g e s o v e r o t h e r c h e m i c a l s in a t t e m p t i n g to s t u d y t h e m e c h a n i s m o f d i f f e r e n t i a t i o n in H L 6 0 cells. Its a c t i o n in i n h i b i t i n g h i s t o n e d e a c e t y l a s e a p p e a r s t o b e t h e Critical e v e n t in i n i t i a t i n g d i f f e r e n t i a t i o n a n d this e f f e c t is r e a d i l y r e v e r s e d by w a s h i n g t h e cells t o r e m o v e b u t y r a t e . T o assess t h e a c t i o n o f this a g e n t o n H L 6 0 w e t r e a t e d c u l t u r e s o f H L 6 0 cells (10 ~ c e l l s / m l ) w i t h s o d i u m b u t y r a t e , r e t i n o i c a c i d , T P A o r n o r m a l saline. T h e c u l t u r e s w e r e h a r v e s t e d a f t e r f o u r days, counted and cytocentrifuge smears prepared and stained with MGG. The results are p r e s e n t e d in T a b l e 1 a n d , as e x p e c t e d f r o m p r e v i o u s l y p u b l i s h e d r e p o r t s , r e t i n o i c a c i d i n d u c e d d i f f e r e n t i a t i o n t o m o r e m a t u r e g r a n u l o c y t i c cells, w h e r e a s T P A i n d u c e d d i f f e r e n t i a t i o n to cells r e s e m b l i n g m a c r o p h a g e s , S o d i u m b u t y r a t e , at c o n c e n t r a t i o n s o f 0 . 1 - 1 m M , i n d u c e d c h a n g e s to cells w h i c h , as in t h e c a s e o f T P A , a p p e a r e d t o b e

30

ANDREW W. BOYDand DONALDMETCALF

monocyte/macrophage in type. However, in contrast to TPA-treated cells, butyratetreated cells were somewhat smaller and largely non-adherent. Virtually 100070 resembled monocytes rather than macrophages. These changes are illustrated in Fig. 1. Figure I(A) shows untreated cells of which over 95070 show blastic features and non-specific cytoplasmic granulation characteristic of promyelocytes. Figures I(B) and I(C) show differentiation to monocytic cells in butyrate-treated cultures which have either retained (B) or lost (C) blastic features.

Sodium butyrate-induced differentiation is not due to selective survival of a subpopulation o f cells A feature of HL60 cultures is the capacity of a small fraction of cells to differentiate spontaneously into either granulocytic or monocytic cells, although in control cultures these cells never exceeded 5070 of the total cell count (Tables 1 and 2). Thus, it appeared possible that the 'inducers' of differentiation might be acting, in a trivial manner, by selectively allowing survival of one or other groups of these spontaneously differentiated cells. To test this possibility we performed a time-course analysis of butyrate treatment with absolute cell counts at each time point. Since in initial experiments, 0.4 mM sodium butyrate induced maximal differentiation, this concentration and a slightly lower concentration (0.1 raM) were chosen for testing. The results of a typical experiment are shown in Table 2. It is evident that in both control and butyrate-treated groups, the cell counts initially increased, but after two to three days the number of cells in the butyrate-treated cultures reached a plateau whereas control cultures continued to increase in an exponential manner. Decreased proliferation in butyrate-treated cultures was paralleled by a progressive increase in the proportion of monocytic cells detected by differential cell counts. The possibility that selection accounted for the increasing population of monocytes, was therefore excluded by the cell count data. For example, at day 3 of treatment with 0.4 mM butyrate 97070 of cells were monocytic or monoblastic compared with less than 5070 initially, but the cell count had actually increased 50070 during that period. Thus in the experiment shown, the absolute number of monocytic cells increased 30-fold during this culture interval. After longer culture intervals, the cell counts decreased as a result of cell death. That this fall in viability was not an inevitable consequence of the differentiation process was shown by washing day 4 butyrate-treated cells twice through FCS to remove butyrate then reculturing them in liquid culture medium. These cultures were able to be maintained for at least four weeks with evidence of only slowly reducing cell viability and continued monocytic morphology throughout that period. Butyrate-treated HL60 cells have phenotypic characteristics of normal monocyte/macrophage lineage cells While butyrate-treated HL00 ceils morphologically resembled monocytic cells, it may be argued that such alterations are artefacts of a process leading eventually to cell death but not representing genuine monocytic differentiation. One way to examine this question was to determine whether these cells could develop functions not present in the control cells, and which implied de novo expression of genetic material consistent with the development of a monocytic phenotype. Several, functional characteristics and surface markers were tested (Table 3) and compared between HL60 cells treated with sodium butyrate, TPA and retinoic acid as well as with control cells. From the data in Table 3, it is evident that butyrate-treated cells, like those treated with TPA, showed a pronounced increase in expression of non-specific estcras¢, phagocytosis and OKM l-positivity, all of which are features of normal monocytes. The expression of these new functions suggests, not passive degenerative events, but an active process of differentiation closely akin to that seen in the normal cells of the myelomonocytic lineage. The OKT9 marker declined,

31

Differentiation induction by sodium butyrate in HL60 cells TABLE |. THE EFFECT OF INDUCERS OF DIFFERENTIATION ON H L 6 0 CELLS

Addition Promyelocyte

Differential cell count Myeioid Monoblastic cells

Monocytes

--

97

2

0

1

Retinoic acid 0.1 p.M Retinoic acid 1 IxM TPA 16 nM Butyrate 0.1 mM Butyrate 0.4 mM Butyrate 1 mM

63 47 7 57 0 0

32 45 6 16 0 0

2 3 25 10 23 0

3 5 62 17 77 100

Cell viability 96 89 67 59 88 72 36

HL60 cells (10'/ml) were cultured for four days in the presence of the inducer, after which the cells were washed, counted and cytocentrifuge smears prepared. Smears were stained with May-Grunwald-Giemsa and a differential count of 200 cells was performed for each group.

TABLE 2. MONOCYTIC DIFFERENTIATION OCCURS OVER FOUR DAYS AND CANNOT BE EXPLAINED BY SELECTION OF A MINOR SUBPOPULATION

Addition

Butyrate 0.1 mM

Butyrate 0.4 mM

Time of exposure (days)

Differential count Promyeloeytes

Myeloid forms

Cell count/

Monoblasts

Monocytes

1

96

2

1

1

2 3 4 5 7 1 2 3 4 5 7

96 97 98 96 95 92 86 84 60 40 0

0 1 0 2 2 4 4 8 28 16 0

3 2 ! 1 2 3 5 5 8 25 0

1 0 1 1 1 1 5 3 4 19 100

1

76

1

19

4

2 3 4 5 7

10 3 0 0 0

0 0 0 0 0

36 41 0 0 0

54 56 100 100 100

Cell count initially* 1.8 3.2 5.0 7.8 11.0 21.0 1.7 2.3 2.6 2.8 1.2 0.6 1.3

1.6 1.5 1.3 0.6 0.17

Cells were cultured for varying periods with sodium butyrate, washed, counted and smears prepared. *Total cell count at each time point divided by the starting cell count.

rather than increased during differentiation. This antibody has been shown to bind to the t r a n s f e r r i n r e c e p t o r [16] a n d t o b e a m a r k e r o f p r o l i f e r a t i o n w h i c h r e d u c e s g r e a t l y o n H L 6 0 cells a f t e r e x p o s u r e t o D M S O [1, 22]. T h e p r o g r e s s i v e f a l l o f t h e l e v e l o f O K T 9 a f t e r b u t y r a t e t r e a t m e n t is in k e e p i n g w i t h t r a n s i t i o n t o a n o n - d i v i d i n g s t a t e .

32

ANDREW W. BOYD a nd DONALD METCALF TABLE 3. BUTYRATE-TREATED HL60 CELLS DEMONSTRATE FEATURESCHARACTERISTICOF NORMAL MONOCYTES

Percentage of cells exhibiting specified property Untreated Sodium butyrate Retinoic acid TPA 0.4 mM 0.1 gM 16 nM

Test

Morphology

Promyelocyte

Monocyte (greater than 95%)

Granulocytic (70%)

Adherent macrophage

00%) Plastic adherence Non-specific esterase*

Less than 1 2 (:i:)

7 96 (+ to + + +)

Less than 1 4 ( 4- to +)

Over 90 87 (+ to + +)

Monoclonai antibody staining:t --

FMC 10 FMC 13 FMC 17 FMC 34 OLM 1 OKT 9 Phagocytosis: I Bacteria Opsoninized sheep red blood cells

2

2

3

5

73 27 0 53 8 97

31 27 5 84 91 0§

23 16 0 63 13 NT

NT:~ NT 4 NT 86 NT

11

67

23

94

<1

43

17

37

*Cytoeentrifnge smears stained for non-specific esterase were scored as negative, equivocal ('+'), weakly positive (+), moderate positive (+ +) and strongly positive (+ + +), based on positive cells (monocytes) in stained smears of normal peripheral blood. tCells (10") were held on ice for 60 rain with each monoclonal antibody. The cells were washed through FCS and a second layer antibody (fluoresceln-conjugated Fab '2 fragments of sheep anti-mouse antibody) added at 1:20 dilution. Cells were analysed on the fluorescence activated cell sorter (FACS) and percentage positive cells obtained with reference to control profiles. :[:NT: not tested. §FACS analysis showed most to be weakly positive relative to control antibody but all were negative by fluorescence microscopy. UFor both methods cytocentrifuge smears were stained with MGG and the cells examined for intrace!lular parasites or sheep red blood cells, respectively.

Differentiation induction is coupled to loss of clonogenic potential in HL60 cells O n e f e a t u r e o f n o r m a l m y e l o m o n o c y t i c d i f f e r e n t i a t i o n is a r e d u c t i o n a n d e v e n t u a l loss b y cells o f the c a p a c i t y to p r o l i f e r a t e a f t e r a c q u i r i n g t h e m a t u r e p h e n o t y p e . T h e d a t a p r e s e n t e d in T a b l e 2 a n d the e v i d e n c e r e l a t e d to the O K T 9 m a r k e r suggested t h a t this m a y a l s o be o c c u r r i n g in b u t y r a t e - t r e a t e d H L 6 0 cultures. T o e x a m i n e this m o r e critically a n d to d e t e r m i n e if this was irreversible we c u l t u r e d H L 6 0 cells with 0.1 o r 0.4 m M s o d i u m b u t y r a t e f o r v a r y i n g intervals, in liquid culture. T h e b u t y r a t e was t h e n r e m o v e d by c a r e f u l l y w a s h i n g the cells twice t h r o u g h u n d e r l a y e r s o f F C S . T h a t this p r o c e d u r e a d e q u a t e l y r e m o v e d the b u t y r a t e was p r o v e d in t w o ways. Firstly, H L 6 0 cells were pulsed on ice with 0.1 m M s o d i u m b u t y r a t e , w a s h e d as d e s c r i b e d a b o v e , then c u l t u r e d in a g a r . This p r o c e d u r e h a d no effect o n c o l o n y n u m b e r , size o r m o r p h o l o g y w h e n c o m p a r e d with c o n t r o l cultures. S e c o n d l y , c o n t r o l cells a n d cells t r e a t e d with b u t y r a t e (0.4 m M ) for f o u r d a y s were w a s h e d , the two p o p u l a t i o n s m i x e d in v a r y i n g p o r p o r t i o n s a n d then c u l t u r e d in a g a r . In this case, c o l o n y n u m b e r s , size a n d m o r p h o l o g y were as e x p e c t e d f r o m s i m p l e m i x i n g w h e n c o m p a r e d with the results f r o m c u l t u r e o f either p o p u l a t i o n alone.

FiG. I. H L 6 0 ceils at various Stallesof butyrate-indumd differentiation.Panel A ~ untreated H L 6 0 cells.Panel B ~ cellsafter two days butyrate treatment; ~ranules have been lostbut the cells retain blast-like nuclei. Panel C ~ day 4 of butyrate treaUnent.

Ftc 5. Photomicrolffaphs of untreated HL60 cells sorted on the basis of OKMI staining. Panel A shows the n e p t i v e fraction - - uniform, blur-like cells. Panels labelled B show a representative selection of OKMI- positive cells.

Differentiation induction by sodium butyrate in HL60 cells

35

Cultures o f cells which had been pretreated with b u t y r a t e were assessed after 10 days. The n u m b e r s o f colonies (as a percentage o f control) at each time point are shown in Fig. 2. It was evident that b u t y r a t e t r e a t m e n t resulted in a progressive reduction o f c o l o n y n u m b e r s to virtually zero after f o u r to seven days, A striking feature o f these cultures was that persisting clonogenic cells gave rise to colonies that were similar in size and a p p e a r a n c e to control colonies. T o test further that these colonies were unaltered, individual colonies were recloned in agar once. T h e n u m b e r o f resulting ' s e c o n d a r y ' colonies was not significantly different f r o m that generated by colonies arising f r o m untreated H L 6 0 cells.

t I

Ifl

IIi O

.o

£ O O~ O £

2

t

t

\

I

I I

I I I I I

0

I

\ \

0 1 2 3 4 5 6 7 Duration of pre-lreatmer~t with Butyrate (days)

F,(;. 2. Clonogenic HL60 cell (colony) number compared with duration of pretreatment with butyrate. Cells were exposed to sodium butyrate 0.1 mM( @ ) or 0.4 mM ( [] ) for varying times (shown on the horizontal axis). After washing away butyrate the cells were cultured in agar and colonies enumerated after 14 days. Colony numbers are expressed as percentage of colony numbers in cultures of untreated HL60 cells for each time point.

Butyrate induction o f differentiation is not a uniform process in all cells The survival o f some c o l o n y - f o r m i n g cells in the presence o f butyrate, which, as assessed by their capacity to proliferate following r e m o v a l o f butyrate, were identical to control cells, suggests some all-or-none process which at r a n d o m m a y spare some cells after even one week o f treatment. In other words, the progressive fall in clonogenic cells suggests a stochastic element to this 'switching' process. T o examine this possibility m o r e closely, H L 6 0 cells were cultured in agar either in the c o n t i n u o u s presence or in the

ANDREW W. BOYD and DONALD MET(_mALF

36

absence of butyrate (0.1 mM). At successive time intervals, cultures were examined and the total number of 'cloned cell units' (single cells, clusters and colonies) counted. For each clonal unit, an estimate was also made of the number of cells present. The results of this study are shown in Fig. 3. Comparative observations of control and butyrate-treated cultures could not be extended beyond four days of incubation since accurate assessment of cell number of cells in the enlarging control clones became impossible after that time. At day 1 (Fig. 3, panel A) approx. 70% of the cells had undergone division in both control and butyrate cultures. The only difference noted was that whereas in control cultures the paired daughter cells remained in close contact with one another, in the butyrate-treated cultures the cells of some daughter pairs had separated and one or both cells appeared somewhat smaller than control cells. By two days (Fig. 3, panel B), although the total 'cloned units' (Fig. 3, panel D) were the same in both groups, many butyrate-treated clones had failed to progress beyond the two-cell stage, while the majority of control

O.

-~0 -120 I00 1

2 3 I~lm of Culture

4

-~.--~__#-I

-30 -20

Lml--L I ,.-.,

i

.

F- ' - - - J

--.J'--J

Cells per Clone

FIG. 3. HL60 colony size in butyrate-treated cultures. HL60 cells were cultured in agar with or without sodium butyrate. The cultures were examined after one, two arid four days. The distribution of cell number per clonal unit is displayed (Panels A-C) for each time point for control (broken line) and butyrate-treated (solid line) cells. Panel D (insert) shows the total viable clones (per 200 input cells) for control ( - A --) and butyrate-treated ( - - o - - ) cultures.

Differentiationinductionby sodium.butyratein HL60cells "'~o

37

I

(a)

Z,

lO0 200 Forword t~jht scot~er

[~0

I

I

~ 0

iO

I 2 Forwon01 Light scotter

.00

~000

iO

FLuorescence I

I IIIllll

I I Illllll

1

I

IIIll|

Jl I0 I00 Ftuorescence

~0

~000

FLuorescence I

Illlllll

I IIIIllll

I

|111111

I000

I0 I00 Ftuorescence

IOOC

FiG. 4. Fluorescence-activated cell sorter (FACS) profiles of control and butyrate-treated cells. Panels a-c are control cell profiles and panels d-f butyrate-treated cell profiles. Panels a and d depict the size distriction (0 ° light scatter) for control and butyrate-treated cultures respectively. Panels b and e show fluorescence distributions of OKMI antibody staining and panels c and f show FMCIO antibody staining.

clones now contained three or four cells. At four days (Fig. 3, panel C) this trend was even more apparent, but it should be noted that the growth of a subset of butyrate-treated clones still matched that of control clones (as expected from Fig. 2). To test whether this represented an intrinsic difference in this subset of clonogenic cells some persisting colonies were expanded in liquid culture and re-tested. Failure to respond to butyrate was not evident and a similar heterogeneity of responsiveness was noted. The smaller average size of butyrate-treated clones at early times was not due simply to a prolongation of the cell cycle time since those clones failed to increase in size after 7-10 days of observation. It was also apparent that by day 4 of incubation, cell death was occurring in the butyratetreated cultures, leading to a fall in total viable clone numbers, as shown in Fig. 3, panel D. These data indicate that the presence of sodium butyrate does not affect the capacity of cells to survive in agar medium for at least two days and even allows most to pass through several successive divisions before responding to a stimulus which halts proliferation. Control cell cultures are heterogeneous in their capacity to proliferate in a cional manner

In the light of the above results, it was of interest to determine whether the spontaneously differentiated cells found in control suspension of cultures of HL60 cells also exhibited an irreversible loss of proliferative potential. One cell surface marker which clearly identified differentiated cells during fluorescence-activated cell sorting (FACS) of untreated cultures was positive OKM1 antibody staining. Staining of control and butyrate-treated cultures with OKM1 antibody is shown in Fig. 4, the top panels (a, b, c) are control cells and the bottom (d, e, f) are of butyrate-treated cells. From left to right they depict cell number on the vertical axis, against on the horizontal axis zero degree light scatter which correlates in the first panel with cell size (a, d), in the second panel with

ANDREW W. I~)YD and DONALDMETCALF

38

fluorescence of OKMl-labelled cells (b, e) and finally in the last panel with fluorescence of FMC 10-labelled cells (c, f). From the data in the middle panels (b, e), it is evident that only a minor subpopulation of control cells (about 5 °70) was positive with OKM 1 antibody (panel b), whereas virtually all butyrate-treated cells were positive (panel e), Some change also occurred with FMC 10, but the differences between normal and butyrate-treated cells were less obvious. The changes in OKM1 were also demonstrated by SDS-PAGE of immunoprecipitates (with OKM 1) of I'25-1abelled cell surface OKM 1 antigen. In controls the antigen was virtually undetectable whereas after two days exposure to sodium butyrate a strong band was evident. Untreated HL60 cells labelled with OKMI antibody were sorted into high fluorescence and low fluorescence subpopulations, and differential cell counts and agar cultures performed. These two populations are shown in Fig. 5, the low fluorescence population (A) being almost entirely blast cells, whereas the smaller high fluorescence population (B) consisted of a mixture of more mature myeloid and monocytic cells. The differential counts of the two populations are shown in Table 4 with the results of culturing the cells in agar medium. These results showed that the OKMl-positive population appeared to contain a TABLE 4. COMPARISON OF HL60 CELLS HAVING HIGH AND LOW OKMI ANTIBODY LABELLINGON THE CELL SURFACE

Cell fraction

% of total

Unsorted

Differential count Colonies Promyelocyte Myeloid Monocytoid per 300 cells

100

High OKMI staining

Low OKMi staining

7 93

97

2

1

143 -6 7

28

61

11

11 + 1

99

1

0

84 -6 6

Control HL60 celts were treated witk OKM 1 antibody followed by fluoresceinated Fab '2 fragments of'a rabbit anti-mouse ig antibody. After whshing the cells were sorted on the fluorescence-activated cell sorter on the basis of fluorescence. TABLE 5. SPONTANEOUSLY-DIFFERENTIATINGHL60 CELLS EXHIBITREDUCEDCLONOGENICITY

Fraction

Differential cell count

%

Colonies per 300 cells

Promyelocytes

Myelocytes

Monocytoid

94 98 64

4 2 20

2 0 16

(A) FACS exp.*

Unsorted Large

Small

I00 88 12

126 -4- 7 142 "+- 9 27 ± 5t

(B) Density aradient separation (Percoll):l:(lessthan) 1.06 1.077 1.09

Unfractionated

82 13 5 I00

98 96 90 96

2 3 8 3

0 I 2 I

129 120 74 133

-I- 7 ± 5 4- 8t ± 5

*Cells were sorted on the FACS on the basis of low angle light scatter which is proportional

to cell size. t p < 0 . O I compared with unsorted population (t-test for paired variates). :I:A three-step density gradient was established by carefully layering of Percoll suspensions of

the indicated densities. The cell suspension was layered on top and then centrifuged for 30 min at 800 g.

Differentiationinductionby sodiumbutyratein HL60cells

39

lower frequency of cionogenic cells. However, it should be noted that the cloning efficiency of the low OKMI staining blast cell population was lower than that of unsorted cells. This may have been due to damage to some cells during passage through the cell sorter or due to the effects of the bound OKM 1 antibody. To check this latter possibility we made use of another feature of these cells, that is, that like the butyrate-treated cells (Fig. 4, left-hand panels), they were smaller than the average of all control cells. Sorting on the basis of size alone, with no labelling procedure, resulted in a far less adequate separation of the differentiated cells than using OKM 1 labelling, but in this case the sorted cells, when all fractions were mixed, retained their clonogenicity. It was again evident that the smaller differentiated cells exhibited reduced clonogenicity (Table 5, part A). Finally, the possibility of density gradient separation was explored using a three-step Percoll gradient to obtain three-cell fractions. This achieved a rather poor level of enrichment of morphologically-differentiated cells but as in the FACS separation based on size, the fraction containing the highest proportion of differentiated cells also contained the lowest frequency of clonogenic cells (Table 5, part B). It is also apparent that the cloning efficiency is reduced out of proportion to the number of identifiably differentiated cells. This suggests that loss of clonogenicity may precede expression of differentiation markers (as occurs with butyrate).

Differentiation-induction in HL60 is linked to cell cycle status The induction by sodium butyrate of a precise change such as monocytic differentiation is puzzling, in that the action of butyrate might be anticipated to be quite non-specific and to affect the entire genome. Why then do HL60 cells become monocytes, rather than erythrocytes or muscle cells? The limited ability of untreated HL60 cells to differentiate spontaneously may hold the key to this question since this property implies the partial activation of this pathway of differentiation. Thus if an HL60 cell could be perturbed during a critical part of the cell cycle, it might shut-off the option of dividing again and follow one specific, already active, pathway of differentiation. One possible consequence of such a process might be that the cells, once committed to differentiation, might become arrested at a specific phase of the cell cycle rather than being arrested at random throughout the cycle as might be expected in a one-step toxic process. To test this, we stained control and butyrate-treated HL60 cells for 90 min at 37°C with Hoechst 33342 stain which is taken up into DNA in a quantitative manner. The fluorescence profiles (on the FACS) of control and four-day butyrate-treated cells are shown in Fig. 6. The control cells showed the typical pattern of a dividing cell population with a prominent initial peak (G1 and early S phase) containing about 50°70 of the cells. The remaining 50070 of the cells contain more DNA and are in late S, G2 and M phase. The butyrate-treated cells showed an altered profile with fewer than 10070 of the cells in the second peak. However, this proportion of cells containing increased amounts of DNA appeared to contradict the previous cloning data (Fig. 2) which suggested that after this duration of butyrate-treatment, fewer than 1°70 of cells would have remained clonogenic. For this reason, the butyrate-treated cells in the two peaks were sorted and the cell morphology examined. The discrepancy was explained by the data obtained, since nearly all the cells in the second peak of butyratetreated cultures were multinucleate (usually binucleate) and of 500 ceils counted, only one mitosis was observed. By contrast, approx. 5% of cells in the second peak of control cultures contained mitotic figures. Thus we concluded that most butyrate-treated HL60 cells were arrested, on the basis of DNA content, in the G1 phase of the cell cycle. This raised the question of whether the morphological and cell surface marker changes are related to cell-cycle status rather than a discrete pathway of differentiation. To answer this, HL60 cells were sorted into three fractions, the G1-S1 fraction being the cells to the left of the maximum of the G1 peak, S cells being taken from the end of the first peak, to

ANDREW W. BOYD and DONALD METCALF

40

I

I

!

(a)

I

(b)

Z00 "

.'.." :.

I

0

I

I

I00

200

IO0

Forword tight ~.otter 8oo

T

I

200

Forv~'d Ligl~ ~otter r-'--

!

T"---

(d)

t m o

.,J

200

IOO

tOO

200

Ftuocelce~ce

FiG. 6. FACS analysis of HL60 cultures treated with Hoechst 33342 stain. Panels a and b depict scatter diagrams of DNA content (fluorescence) vs cell size (forward light scatter) for untreated (Panel a) and butyrate-treated culture (Panel b). Panels c and d show cell number vs DNA content (fluorescence) for untreated (Panel c) and butyrate-treated cultures (Panel d).

the inflection of the G2-M peak and finally the whole G2-M peak. The morphology of the majority of cells in all fractions was promyeloc~ic and EACS-analysis of the levels of OKT9 and OKMI (which were considerably decreased and increased respectively on butyrate-treated cells) showed only minor fluctuations which were in keeping with the increase in cell size during progression through the cycle. The final state of butyrate-treated cells is cell-cycle restricted but it was also of interest to determine if the trigger for this action was also limited to one phase of the cycle. Untreated HL60 cells were sorted into two fractions, one including all cells to the left of the T A B L E 6 . RESPONSE OF

HL60

CELLS IN DIFFERENT C E L L C Y C L E

STAGES OF BUTYRATE

TREATMENT

Cell cycle fraction*

Colonies per 400 cells Untreated 15 h pre-treatmertt with butyrate (0.3 raM)

Sz-G2-Mpeak GI-S, peak Unsorted

174"+"10 207 =1= 16 139::t=23

90-t- 7t 219-t- I1 124 =1= 16

*Cells were stained with Hoechst 33342 dye for 90 min at 37°C. After washing, the cells were held on ice in a container sealed against the light prior to sorting on the FACS on the basis of fluorescence (DNA content). tp<0.01 compared with untreated S2-G2-M phase cells (t-test for paired variates).

Differentiationinductionby sodiumbutyratein HL60cells

41

mid-point of the down slope of the first peak. The second fraction included all cells to the right of the mid-point of the plateau between the two peaks. These two fractions were then subjected to butyrate treatment for 15 h, after which the cells were washed and cultured in agar medium. As is shown in Table 6, the effect of butyrate in suppressing clonogenicity was more evident on the cells that were initially in the S2-G2-M phases than those in the GI-S1 phase. Considering that HL60 cells have a long S phase (approx. 14 h) representing about 60°70 of the total cell cycle time [13] this suggests that only the second peak cells would have divided during the period of exposure to butyrate and hence that the trigger for differentiation is very late in the cycle or early in G1 since these are the only phases not strongly represented by the GI-SI fraction during the culture period.

DISCUSSION This report has shown that sodium butyrate induces monocytic differentiationin H L 6 0 cells(Table I and Fig. I). W h e n added to cultures at a concentration of 0.4-0.6 raM, these changes had affected nearly all the cellsafter four days but viabilitywas reasonably well preserved (Table 2). Prolonged treatment resulted in cell death but removal of butyrate (by washing) at four to seven days resulted in persistenceof viable, differentiatedcellsfor up to four weeks. The possibilitythat these changes represented differentialsurvival of the small spontaneously differentiated population in untreated H L 6 0 cultures was excluded by examination of cell counts (Table 2). The cells appeared morphologically to be monocytic, but this was also checked by testing butyrate-treated ceUs for induction of other monocytic features. The results of these studies (summarized in Table 3) showed that the butyrate-treated cells had functional, cell surface marker and cytochemical features of mature monocytes. The expression of these features imply metabolically active differentiation rather than manifestations of toxicity and are in keeping with the idea that HL60, while being a tumour, preserves the capacity to undergo nearly normal differentiation along the monocyte/ macrophage pathway. Also in keeping with this notion was the loss of proliferativecapacity which occurred in tandem with differentiation.This was manifest by the loss of transferrinreceptors (OKT9 binding) to the cell surface (Table 3) and the inability of differentiated cells to form colonies in agar (Fig. 2). Both of these processes were progressive over about four days and, ifanything, slightlypreceded the manifestations of monocytic differentiation.This is in accord with previous observations of DMSO-induced differentiation of H L 6 0 cells [12]. The small (less than 5°7o) population of spontaneously differentiated cells in untreated HL60 cultures was also examined. The most conspicuous marker of differentiation induced by butyrate treatment was the increase in the OKM1 marker. Using this marker on control cells, we showed that the OKMl-positive population from untreated HL60 cultures consisted of approx. 30070 blast cells but the remaining subpopulation was a mixture of differentiated myeloid (60070) and monocytic 10070 cells (Fig. 3 and Table 4). This population was shown to have a reduced cloning efficiency relative to the low OKM1 population which contained greater than 99070 blast cells. Two other parameters (cell size and cell density) were used to obtain a less satisfactory enrichment of spontaneously differentiated cells. The result of agar culture (Table 5) in each case was again consistent with the idea that the spontaneously differentiated cells, like cells induced to differentiate by sodium butyrate, had lost clonogenic potential. The mechanism of induction of differentiation by sodium butyrate was examined in two ways. First, agar cultures were established with or without sodium butyrate, and the cultures examined each day to assess the distribution of clone size. These results (Fig. 4) showed that for 24 h no difference was evident, suggesting that, at least initially, division

42

ANDREWW. BOYDand DONALDMETCALF

rate was unaffected, but thereafter butyrate-treated clones lagged behind controls, most being arrested at the one- to four-cell stage. However, when butyrate was removed from cultures, the surviving clonogenic cells produced normal-sized colonies (Fig. 2) and normal numbers o f secondary colonies after replating o f individual colonies. Thus a minority o f butyrate-treated clonogenic cells continue to exhibit the characteristics of control HL60 cells. This suggested that the process of differentiation-induction involved an all-or-none process. The observation that the majority of butyrate-treated cells appeared to divide in the first 24 h at the same rate as untreated cells taken together with the rapid onset o f the effects o f butyrate on enzyme activity [3, 6, 29, 36] suggested that a chain of events had been initiated by butyrate rather than a single-step process. That all differentiated cells were arrested in GI rather than at random throughout the cell cycle is also in keeping with this idea (Fig. 5). In the light o f this observation, the possibility that the point at which clonogenicity was lost might be cell-cycle restricted was tested. Untreated HL60 cells were sorted on the basis o f DNA content into two fractions, those in GI and early S1 in one fraction and those in G2-M in the other (Fig. 0c). The two populations were treated with butyrate for 15 h then cultured in agar. Only the G2"-M cells, which would have divided and entered G1 during that time showed evidence of differentiationinduction and loss o f clonogenicity. The known cycle time of HL60 cells suggests that very few o f the Gl-early S phase cells could have reached G2-M, let alone G1, and these cells showed no response to butyrate. These findings are at variance with those observed when DMSO was employed as an inducer [30], in that case no cell cycle restriction was observed. This may reflect a slower reversal of DMSO-induced effects compared with butyrate which would blur the restriction to a single phase of the cell cycle. Several lines of evidence have implicated the GI phase as a potential control point of the rate o f cell proliferation [7, 28, 33] and on theoretical grounds [4] this control phase is likely to consist of several individual, successive and stochastically-variable events. That a discrete phase o f the cell cycle might also be linked to differentiation-induction has been supported in the study o f pro-adipocyte differentiation [32]. With this background in mind and considering the evidence presented in this report it would appear that buty.rateinduced differentiation is an all-or-none event initiated during a particular phase of the cell cycle which is likely to occur in relation to cell division, either late in the cycle or in GI. The phenomenon o f spontaneous differentiation in untreated cultures added to cell kinetic data on control populations [13] suggests that the option of differentiation into a non-dividing state is usually available to HL60 cells, but that butyrate (and other inducers) alter the probability o f a cell entering this pathway (with associated irreversible loss of the capacity to divide) rather than continuing to divide. This model of differentiation would explain why butyrate, with its apparently non-specific mode of action results in a specific pattern o f differentiation in a given cell line. In other words, the process initiated by butyrate may hinge on molecular mechanisms present in all dividing cells, both malignant and normal. HL00 cells are a convenient model in which to attempt to dissect out these nuclear mechanisms, which might reasonably be expected to operate in all turnouts to a varying degree. Knowledge o f these processes may help us to understand the events o f normal differentiation and suggests new means of controlling tumour cell growth. AcknowledgementswThe authors wish to acknowledge the excellent technical assistance of Ms. Elizabeth Viney. We also wish to thank Dr Frank Battye and Mr Mark Cozens for their help in experimentsinvolvingthe FACS, and Ms. Sue O'Mahoney for performing special stains.

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