Kinetics of histone H1 ° accumulation and commitment to differentiation in murine erythroleukemia cells

Experimental Cell Research I52 (1984) 449-458

Kinetics of Histone to Differentiation H. BEVERLEY

HI” Accumulation and Commitment in Murine Erythroleukemia CeNs OSBORNE’ and AGNES CHABANAS’

‘Laboratoire de Biologie Molkculaire et Cellulaire, 2(ER 199 du CNRS), Departement de Recherche Fondamentale, Centre d’Etudes Nucltaires de Grenoble, 85X, 38041 Grenoble, Cedex, France

The accumulation of histone H1° (also denoted IP 25) in murine erythroleukemia cells, induced to differentiate with hexamethylene his-acetamide, was shown to precede by 15-20 h the appearance in the culture of cells irreversibly committed to differentiate. In addition the rates of accumulation of H1° and of committed cells vary in a similar manner with the HMBA concentration. Flow microfluorimetric analysis demonstrated that the accumulation of H1° did not occur simultaneously in all the cells. This accumulation of histone H1° was initiated first in cell in the G2 phase of the cell cycle and subsequently in the cells situated in all the phases of the cell cycle.

The differentiation of murine erythroleukemia (MEL) cells can be induced by a wide variety of different agents and the cellular modifications associated with this differentiation have been well characterized (for review see [l]). However, little is known about either the functional relationship between these modifications or their necessity for the continuation of the differentiation program to the point at which it becomes irreversible (termed commitment). One approach to this problem has been to inhibit certain metabolic functions [2-4] or to modify the environment of the cells [5] and then to determine whether the induced differentiation has also been inhibited. However, this approach is only applicable if inhibitors of the induced cellular modification being studied are known. Amongst the modifications which appear in MEL cells prior to the commitment event, the accumulation of IP 25 first reported by Keppel et al. [6] is of particular interest. Zlatanova et al. [7] showed that IP 25 was identical with a subfraction of histone Hl-Hl’. This subfraction of histone Hl, which accumulates in the nuclei of a large number of different cellular systems when they are induced to differentiate [8], has often been correlated with a loss of proliferative capacity of the constituent cells [P-11]. In addition the presence of histone H1° has been positively correlated with the hormonally dependent functional activity of cells from several rat and mouse tissues [ill. In MEL cells the accumulation of histone H1° appears to precede the commitment event [6], although the moment at which this accumulation is initiated has not been determined. Several observations suggest that the presence of H1° may be correlated directly with the modifications necessary for the commitment event to occur. In at least one DMSO-resistant clone, no histone H1° was synthesized Copyright @I 1984 by Academic Press, Inc. All rights of reproduction in any form nxerved 0014-4827r%4503.00

450 Osborne and Chabanas [6]. Furthermore, hemin, which in certain MEL cell lines induce the synthesis of globin mRNA [12], provokes neither the irreversible commitment to erythrodifferentiation normally observed prior to the synthesis of globin mRNA, nor the accumulation of histone H1° 1131. In the present study use has been made of two characteristics of the induced differentiation itself to obtain information about an eventual causal role for the accumulation of Hl’. Firstly, the rate at which MEL cells become committed to differentiate is a function of the inducer concentration [14]. Since the commitment event is currently assumed to be the result of the sequential accumulation of certain modifications within the cell [ 151, then varying the inducer concentration should similarly affect the rate at which those modifications (necessary for the eventual commitment event to occur) appear in the cell. Secondly, MEL cells must traverse a critical region of the cell cycle (between late Gl and early S phases) in the presence of inducer for cells committed to erythro-differentiation to appear in the culture during the subsequent Gl phase [l&19]. Therefore the initiation of some (but not necessarily all) of the cellular modifications responsible for commitment to erythro-differentiation should also be cell cycle-dependent. Hence two questions have been asked: (a) Is the rate of H1° accumulation dependent upon the inducer concentration? (b) Is the amount of this histone increased first in those cells which were in late Gl or early S phases when the inducer was added to the culture medium? The fulfilment of one or the other of these criteria would imply that the accumulation of HI0 would be either regulated by an event necessary for the subsequent commitment event to occur or would itself be necessary for this event.

MATERIALS

AND METHODS

Cell Culture MEL cells (clone DS 19) were cultured at 37°C in Eagle’s modiEed minimum essential medium supplemented with fetal calf serum (FCS) (15 %) as previously described 1201.Cultures of exponentially growing cells were established prior to use in the experiments described by maintaining the cell concentration between 2x 16 and 1x lob cell/ml, by daily dilution into medium suppkmented with 5% FCS, over a period of 3 days. Differentiation was induced by the addition of HMBA to the culture medium. Benzidine staining [21] and the determination of commitment to erythro-differentiation were performed as previously described [20].

Gel Electrophoretic Analysis of Nuclear Proteins The nuclear fractions of MEL ceils were obtained and the chromatin solubilized as previously described 1221.The nuclear proteins ~nereanalysed on SDS-FACIE (g-15 %) according to the discontinuous buffer system of Laemmli [23]. The gels were fixed with formaldehyde and stained with Coomassie Blue [24]. The gel lanes were traced with a lkansidyne recording microdensitometer and the relative amounts of the different histone HI subfractions estimated from the area under the respective peaks.

ExpCellRes152 (1984)

HI0 accumulation in induced MEL cells 451

I. Kinetics of H1° accumulation and of commitment to differentiation of MEL cells. Exponentially growing MEL cells were cultured with HMBA (4 mM) for various time periods. The nuclei were then extracted, digested with micrococcal nuclease and dissolved in SDS. The relative amount of H1° to total Hl was visualised by electrophoresis of these samples on PAGE in the presence of SDS. After fixation with formaldehyde and staining with Coomassie Blue the relative amount of H1° to total Hl (A) was determined by planimetry of densitometer scans from these gels. This ratio was independent of the amount of material deposited. The proportion of cells committed to erythrodifferentiation (0) at a given time was determined by transferring aliquots of these ceils into medium without inducer. The proportion of cells staining with benzidine, 24 h after the transfer was performed, is equal to the proportion of committed cells in the culture at the time of transfer.

Fig.

Flow Microfluorimetric

Analysis

H1° was titrated by staining with affinity-purified rabbit anti-mouse H1° antibodies followed by a fluorescein-conjugated IgG fraction of goat anti-rabbit IgG (Cappel Laboratories) as previously described [25]. DNA was stained with 4’,6’-diamidino-2 phenylindol dihydrochloride (DAPI, Boehringer) [251. The doubly stained cells were analysed in a FACS IV (Becton-Dickinson), using a dual laser configuration [25]. The DAPI fluorescence was excited at 340-360 nm by a 5W argon laser (full power) and the emitted light was measured on a detector after passage through a 440&O nm band pass filter (no. 70706, Oriel). The fluorescein fluorescence was excited at 488 nm by a second argon laser (output power 500 mW) and the emitted fluorescence was measured on the second detector after passage through a 500-540 nm band pass filter (700716, Oriel).

RESULTS Kinetics of Histone HZ0 Accumulation and of Commitment to Erythro-diflerentiation The accumulation of histone Hl’, after the addition of HMBA to the culture medium of MEL cells, can be easily visualized by electrophoresis of nuclear proteins on polyacrylamide slab gels in the presence of SDS [6]. Histone H1° also accumulates in the nuclei of stationary phase MEL cells in the absence of inducer (unpublished results). Hence, in all the experiments described here the cells were kept in the exponential growth phase for 3 days before the initiation of the Exp Cell Res IS2 (1984)

452 Osborne and Chabanas differentiation process. At various times after the addition of the inducer to the culture medium aliquots of the cell suspension were taken and the nuclear proteins extracted. These extracts were analysed by PAGE and the amount of histone H lo, relative to the total amount of histone HI, was quantified by densitometer scans of these gels. The results, shown in fig. 1, demonstrate that histone HI0 accumulates rapidly during the first 24 h after the addition of HMBA. Thereafter the amount of histone H1° relative to the amount of total histone Hl increases at a slower rate to reach its maximum value 2-3 days after the addition of the inducer. It is important to note that the cell density continued to increase logarithmically throughout this period and that the total amount of histone Hl (Hl-l+Hl-2+H10) was not affected by the presence of HMBA (data not given). The comparison of these kinetics with those of commitment to erythro-differentiation shows striking similarities. The proportion of cells committed to erythro-differentiation in the culture at the moment that the aliquots were taken for electrophoretic analysis were determined and are also shown in fig. 1. These kinetics of commitment to differentiation can similarly be divided into approximately two phases: a rapid phase between 20 and 35 h after which about 70% of the cells in the culture are committed to erythro-differentiation, and a slower phase between 35 and 72 h. At least 90% of the cells in the culture were committed to erythro-differentiation after 3 days of culture with 4 mM HMBA. Efiect of HMBA Concentration on the Rates of HI0 Accumulation and of Commitment to Differentiation of MEL Cells Exponentially growing MEL cells were induced to differentiate with various concentrations of HMBA (2, 3 and 4 mM). After 0, 20 and 40 h of culture, the relative amount of HI0 to total histone Hl and the proportion of cells committed to differentiate was determined for each of these cultures. These time periods were chosen because in the presence of 4 mM HMBA the rapid phase of H1° accumulation occurs mainly between 0 and 20 h, whereas for this same concentration of HMBA the maximum increase in the proportion of committed cells occurs approximately between 20 and 40 h. The results of one such experiment are shown in fig. 2A, B. As expected the kinetics with which committed cells appeared in the culture decreased as the HMBA concentration was reduced (fig. 2B). A similar behaviour was also observed (fig. 2A) for the kinetics with which H1° accumulated in the nuclei of these cells. For each of the inducer concentrations used (2, 3 and 4 mM) the increase between 0 and 20 h in the ratio of H1° to total Hl and the proportion of cells which became committed to differentiate between 20 and 40 h was calculated. These values were used to construct the graph shown in fig. 3. It is important to note that these experiments do not differentiate between an accumulation of HI0 in all the cells or an accumulation in a subpopulation of the cells. Therefore the physiological significance of the linear relationship between these two parameters cannot be ascertained at this point. ExpCellRes152 (1984)

I I

HI’ accumulation in induced MEL cells 453

2A

6

3

a ‘e

.Y

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; E 282 : : i 0

20 Time (hours)

40

Kl

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0 0

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Fig. 2. Effect of HMBA concentration on H1° accumulation and commitment to diierentiation of MEL cells. Exponentially growing MEL cells were seeded at 1x 16 cells/ml in medium containing HMBA 0, 0; 0, 2; l , 3; A, 4 mM. After 20 and 40 h of culture with HMBA the amount of HI0 relative to total Hl in the nuclei of these cells (A) and the proportion of cells committed to differentiate (8) was determined as described in the caption to fig. 1. Fig. 3. Relationship between H1° accumulation and commitment to erythro-differentiation of MEL cells. The increase in the proportion of committed cells which occurs between 20 and 40 h of culture and the increase in the relative amount of HI0 to total Hl between 0 and 20 h, with the various concentrations of HMBA 0,O; l ,2; a, 3; A, 4 mM was calculated from the data given in fig. 2A, B.

Flow Microfluorometric

Analysis of Histone HI0 Accumuiation

The above results do not determine whether the accumulation of histone H1° was initiated in all the cells simultaneously or whether it only occurred in a subpopulation of the cells eventually within a specific phase of the cell cycle. In order to examine this question flow microfluorimetry was used to determine the relative H1° and DNA contents of each cell. After being cultured for various periods with HMBA (4 mM), exponentially growing MEL cells were fixed and labelled with rabbit anti-mouse H1° anti-serum followed by fluorescein isothiocyanate-labelled goat anti-rabbit anti-serum. Labelling with DAPI enabled the relative DNA content to be determined. The results given in fig. 4 show that in the absence of anti-H1° anti-serum (control) no fluorescein fluorescence is observed. In addition cells growing in the absence of HMBA (0 h), contain a basal amount of H1° which increases linearly with the DNA content of the cell. A similar behaviour has been previously reported for Chinese Hamster Ovary cells [25]. The data obtained from the MEL cells cultured for 6 h in the presence of HMBA show that at this time H1° has already started to accumulate in these cells. It should be noted that this accumulation results in an upward dispersion of the data on the ‘dot plots’ and the Exp Cell Res 152 (1984)

454

Osborne and Chabanas

Oh

6h

DNA content

DNA content

lmmunoflunrescence (channel number]

4. Plow microfluorometric analysis of MEL cells at various times after the addition of HMBA. Exponentially growing cells were cultured with HMBA (4 mM) for various periods of time after which ahquots of IO6cells were fixed with 70 % ethanol and the presence of histone H1° revealed by indirect immunofluorescence with rabbit anti-mouse HI0 antibodies followed by fluorescein-conjugated antirabbit IgG antibodies. DNA was stained with DAPI. The flow microfluorometric analysis of these various samples produced a series of two dimensional ‘dot-plots’ of which several are shown in (A) cells cultured for 0, 6 and 8 h with HMBA. The projection of these displays onto the two axes gives the DNA and HlO-specific immunofluoroescence histograms of the whole cell population (8) and (C) respectively. The control cells give the residual fluorescein fluorescence observed when the rabbit anti-mouse HI0 antibody was omitted from the staining procedure.

Fig.

Exp Cell Res 152 (1984)

HI0 accumulation in induced MEL cells 455 Gl (0)

lmmunofluorcscence

20

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time

(hours)

Fig. 5. Cell cycle analysis of H1° accumulation in MEL cells. The fluorescein and DAPI fluores-

cences emitted from the cells stained as described in the caption to fig. 4 was collected in the dual parameter configuration. This allows the data to be displayed in a three-dimensional representation in which the X-axis is DAPI fluorescence; the Y-axis is H1° immunofluorescence and the Z-axis cell number/channel. The insertion of a plane parallel to the Z and Y axes generates an immunofluorescence histogram for the cells whose DNA content is defined by the intersection of this plane with the X-axis. (a) Immunofluorescence histograms obtained from cells cultured for -, 0; ---, 6 h with 4 mM HMBA, for four diierent values of their DAPI fluorescences (the maximum of the Gl and G2+M peaks and for l/3 and 213 the distances between these two maxima denoted early S and late S respectively). The series of histograms for a given DNA content were normalised to give the same surface area. The proportion of cells in the culture, which have increased their H1° immunofluorescence above the basal amount observed at 0 h, can be calculated from these histograms as follows. This quantity is equal to the proportion of the total surface area of a given histogram which is outside (right) of the corresponding histogram obtained from the cells cultured for 0 h with HMBA. (b) The temporal variation of this increased immunofluorescence for the four chosen points in the cell cycle.

broadening of the immunofluorescence histogram. This implies that H1° accumulation does not occur simultaneously in all the cells. These effects are even more evident after 8 h in culture with HMBA. In order to determine whether the increase in the H1° content of the cells is a cell cycle-dependent event, it is necessary to compare at different times after the addition of the inducer, the immunofluorescence histograms of cells with a given DNA content. For such a comparison to be valid the-diRerent histograms for a given DNA content must all represent the same number of cells. This is particularly important for MEL cells as the DNA histograms of the whole cell population E.xp Cell Res IS2 (1984)

456 Osborne and Chabanas is progressively modified after the addition of the inducer (compare fig. 4B; 0; 6 and 8 h). In agreement with the previously published observation of Friedman & Schildkraut [26] an apparent increase in the number of cells in the G2+M phases of the cell cycle was observed before any lengthening of the Gl phase occurred. This effect cannot be due to the presence of bi-nucleated cells, since no cells with DNA contents between 4C and 8C were observable in the ‘dot-plots’. In fig. 5 a are given the immunofluorescence histograms for the cells situated in Gl, early S, late S and G2+M phases of the cell cycle at 0 and 6 h after addition of the inducer. These data show that after 6 h in the presence of HMBA the histograms for all the phases of the cell cycle were displaced to higher fluorescence values, Since the surface area of a part of a histogram is proportional to the number of cells within the chosen fluorescence limits, it is possible from these curves to calculate the proportion of cells with a fluorescence and hence H1° content greater than the initial value. The results obtained from two independent experiments are given in fig. 5 b. They show that the HI’-specific immunofluorescence of the cells in the G2 phase of the cell cycle increased between 2 and 3 h after the addition of the inducer to the culture. Between 3 and 4 h this immunofluorescence also increased in the cells in the Gl and S phases of the cell cycle. For those cells in the Gl phase of the cell cycle a rapid rise in the proportion of cells with increased fluorescence was observed between 6 and 8 h. This was probably due to the mitosis and hence passage into the Gl phase of those cells which had accumulated H1° initially in the G2 phase of the cell cycle. Subsequently the HlO-specific immunofluorescence of the cells in all phases of the cell cycle increased in a similar manner. DISCUSSION The experiments described in this report demonstrate, firstly, that a correlation exists between the accumulation of histone H1° (IP 25) in the nuclei of MEL cells and the proportion of these cells which become committed to erythro-differentiation. Secondly, the initiation of Hl” accumulation was found to occur first in the cells in the G2 phase of the cell cycle and subsequently in all the phases. The accumulation of H1° in the nuclei of MEL cells was initially observed to precede their commitment by 15 to 20 h. However, the kinetic profiles for these two events were very similar. Indeed, in the presence of 4 mM HMBA, about 70% of the total amplitude of both of these events had been attained after an initial rapid phase which lasted about 20 h (for H1° from 0 to 20 h, for commitment from 15 to 35 h). In addition the amount of H1° which accumulates between 0 and 20 h of culture with various concentrations of HMBA was found to be linearly related to the proportion of cells which become committed to differentiate between 20 and 40 h of culture with these same concentrations of HMBA. Flow microfluorimetric analysis of MEL cells stained with HlO-specific antibodies demonstrated that this accumulation of H1° did not occur simultaneously in Exp Cell Res I52 (1984)

Hl” accumulation

in induced MEL cells

457

all the cells of the culture. It is not possible presently to aftirm that the accumulation of H1° is initiated preferentially in those cells which first undergo the commitment event, although the above results suggest that this possibility is not unrealistic. Confirmation of this hypothesis must await the development of a nontoxic probe which will allow cells to be selected from the bulk population several hours before they undergo irreversible commitment to erythro-differentiation. The possible cell cycle dependence of H1° accumulation was also studied by flow microfluorimetry. This analysis demonstrated that the initiation of H1° accumulation was an early event in the differentiation program. The amount of this histone increased in G2 phase cells only 2-3 h after the addition of HMBA to the culture medium. Subsequently (between 3 and 4 h) the amount of histone H1° also increased in the cells in the other phases of the cell cycle. These observations imply that cells situated throughout the entire cell cycle, at the moment that the inducer HMBA was added to the culture medium, could initiate the accumulation of H1° within 4 h. Histone H1° has often been associated with a reduction in cellular proliferation [9-111. Therefore it is interesting to note that in MEL cells the increase in the proportion of cells in G2 phase of the cell cycle, 6 h after the addition of HMBA to the culture medium [26] and fig. 4B, and the already well documented lengthening of the Gl phase, occur after the initiation of H1° accumulation. A pertinent question is the nature of the induced cellular modification(s) which trigger the accumulation of Hl’. From the results presented here two of their characteristics can be deduced. Firstly, these modification(s) will not be restricted to a specific part of the cell cycle and secondly, they should occur soon (less than 2 h) after the addition of HMBA to the culture medium. A consequence of these observations is that the initiation of H1° accumulation may be a useful probe to investigate the regulatory action of early events necessary for the eventual commitment of the cells to erythro-differentiation. The authors wish to thank Dr H. Eisen for the anti-mouse HI0 antibodies and for his helpful comments; Drs J. J. Lawrence and M. Chabre for their useful discussions and M. J. Albert and Mme P. Goeltz for their technical assistance. This work was supported in part by grants from the Wlegation a la Recherche Scientifique et Technique (Comite des Membranes Biologiques and grant no. 81LO726) and also by grants from the CNRS ATP 955158. H. B. 0. and A. C. are members of the Centre National de la Recherche Scientifique (CNRS).

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