Increased histone H1 ° expression in differentiating mouse erythroleukemia cells is related to decreased cell proliferation

EXPERIMENTAL

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RESEARCH

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SHORT NOTE Increased Histone HI O Expression in Differentiating Mouse Erythroleukemia Cells Is Related to Decreased Cell Proliferation JACINT Department Uniuersity

of Biochemistry, at L’H&el-Dieu

BOIX

AND ADOLF

School of Medicine de Q&bee, 11 C&e

The amount of histone Hl” increases relative to other Hl subtypes in terminally differentiated cells, and its expression has been associated with the onset of differentiation. We have studied the kinetics of Hl” accumulation in mouse erythroleukemia (MEL) cells and found that the levels of Hl” reflect the rate of cell proliferation rather than the state of differentiation. This suggests that changes in the relative amount of H lo during MEL cell differentiation are primarily a consequence of cell cycle arrest. 0 1992 Academic Press, Inc.

INTRODUCTION Hl” is a member of the histone Hl family whose expression appears to be characteristic of the state of proliferation and differentiation of mammalian somatic cells [l]. The interest in Hl” resides in its possible involvement in both of these cellular processes. Hl” content increases following proliferation arrest [2-51 and decreases after mitogenic stimulation [6-81, suggesting that the Hl” levels are somehow related to cell growth. Although basal Hl” levels vary with cell origin (e.g., [9, lo]), the highest relative concentrations are found in terminally differentiated tissues with a low rate of cell turnover, where Hl” partially replaces other Hl subtypes [6, 11, 121. These observations have led to the suggestion that Hl” may play a role in the control of cellular proliferation. However, a direct demonstration of the effect of Hl” on cell growth is lacking. In fact, some evidence suggests that the increase in Hl” is a consequence rather than a cause of cell cycle arrest [ 11, 131, and relatively high levels of Hl” do not interfere with the proliferation of certain tumor cell lines [14]. The expression of Hl” has also been correlated with differentiation. Thus, in tissues that depend on hor-

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mones for their function and maintenance, the expression of Hl” appears to depend on the continued presence of the specific maintenance hormone [6]. An increase in Hl” levels also occurs when cells are induced to differentiate in vitro by a variety of inducers [ 15-171. These correlations notwithstanding, an active participation of Hl” in the process of differentiation has not been demonstrated. In fact, there are conflicting reports about the preferential association of Hl” with inactive and active chromatin [ 18, 191. Since differentiation entrains alterations in the cell cycle, it is not obvious from the above results whether the higher expression of HI0 in differentiating cells results from changes in proliferation or is related to differentiation itself. In this report we have sought to distinguish the contribution of each process to Hl” expression using MEL cells which can be induced to differentiate in uitro. MATERIALS

AND

METHODS

Cell cultures. MEL cells (clone 745aJG [20]) were obtained from the late J. A. Connolly and subcloned by the dilution method. A subclone (JB3) that, can be induced to differentiate by hexamethylenebis(acetamide) (HMBA) and dimethyl sulfoxide (DMSO), but not by butyrate, was used in this work. Cells were grown at 37OC and 5% CO? in aMEM supplemented with 20 mM Hepes (pH 7.4) and 10% fetal calf serum. Exponentially growing cultures were diluted to lo5 cells/ml with warm fresh medium and incubated for up to 4 days with or without 4 mM HMBA or 1 mM sodium butyrate. Cell differentiation, as defined by hemoglobin synthesis, was determined by benzidine staining. Eighty to ninety percent of the cells showed positive staining after 4 days when treated with HMBA, as compared to only 510% in its absence or when treated with hutyrate. Less than 5% of the cells were stained with trypan blue at any time during the experiments. Nuclear protein analysis. Acid-soluble nuclear proteins were extracted from isolated nuclei as described [21], or with 5% perchloric acid (PCA) [22], and analyzed by electrophoresis in duplicate 12% polyacrylamide gels containing SDS [23] or acetic acid-urea [24]. One of the gels was stained with Coomassie brilliant blue R and the protein of the other was transferred to nitrocellulose filters (0.2 pm Schleicher & Schuell) [25] and stained with Ponceau’s red. Nitrocellulose blots were reacted with 1 pg/ml of affinity-chroma-

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FIG. 1. (A) Hl” contents of noninduced and induced MEL cells. Acetic acid-urea gel electrophoresis of nuclear proteins extracted with 5% PCA from mouse liver (lane 1) and MEL-JB3 cells induced with 4 mA4 HMBA for 0 (lane 2, 1% benzidine-positive cells) and 4 days (lane 3, 85% benzidine-positive cells). Note the decrease in HMG 1 and 2 during differentiation. (B) Immunological detection of Hl and Hl”. A gel electrophoresis of 5% PCA-soluble nuclear proteins from liver was blotted and stained with Ponceau’s red (lane 1) or reacted with anti-H5 rabbit antibodies and developed with peroxidase-conjugated anti-rabbit antibodies (lane 2).

FIG. 2. Increased Hl” contents of differentiating and control MEL cells. Immunoblots of nuclear proteins separated by SDSPAGE. (A) Parallel cultures of MEL cells were grown in the presence of 4 mM HMBA (lanes 5-8) or in its absence (lanes l-4) for 1, 2,3, and 4 days, respectively. (B) MEL cells were grown 4 days in the presence of 1 mM sodium butyrate (lane 1) or 4 mM HMBA (lane 2).

apparently independent of the presence of the inducer. Since the ratio HlAlcore histones did not vary significantly during the course of the experiments (as judged by densitometry of Coomassie-stained gels; data not shown), the Hl” signal of the immunoblots was measured and expressed relative to the signal of the crossreacting HlA. Figure 3 compares the relative increase in

tography-purified rabbit anti-histone H5 antibodies [26]. These antibodies cross-react with Hl” and to a lesser degree with other Hl’s (Fig. lB, see also [27]). Immunoblots were developed with 0.2 pg/ml of peroxidase-conjugated anti-rabbit antibodies and 3,3-diaminobenzidine with NiCl, enhancement [28]. Immunoreactivity was quantified by scanning photographic negatives of the blots with a Chromoscan 3 microdensitometer (Joyce-Loebl). - 60

RESULTS

Exponential cultures of MEL cells at a density of lo6 cells/ml or less were diluted to lo5 cells/ml and grown with or without 4 mM HMBA. Under these conditions there was no initial lag period and this minimized variations in Hl” content due to changes in growth rate prior to induction by HMBA (see below). To determine the Hl” content of the differentiating cells, nuclear proteins were extracted from controls and cultures exposed to the inducer for l-4 days and analyzed by acetic acid-urea-PAGE. The results shown in Fig. 1A indicate that the MEL cell line used has no detectable Hl” in the noninduced state, and that even after 4 days treatment with HMBA (when 85% of the cells were differentiated) Hl” is barely detectable. To increase the sensitivity of the analysis, we resorted to immunoblotting using rabbit anti-H5 antibodies that cross-react with Hl” and, to a lesser degree, with other Hl’s (Fig. 1B). This method permitted us to detect and to measure Hl” in noninduced MEL cells (Fig. 2). The presence of Hl” in the noninduced cells is in agreement with the presence of Hl” mRNA in these cells (data not shown). The immunoblots of nuclear proteins from HMBAinduced (Fig. 2A, lanes 5-8) and noninduced (Fig. 2A, lanes l-4) cells indicated that Hl” accumulation was

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FIG. 3. Increase in Hl” of MEL cells correlates with decreased rate of proliferation. MEL cell cultures were grown in parallel with (A) and without (B) 4 mM HMBA. Samples were taken at the inclcated periods of time to determine Hl” content, cell density, and extent of differentiation. The relative Hl” increase is expressed as the ratio Hl”/HlA taking that of the 4-day control culture (-HMBA) as reference. Control and HMBA cultures approached the stationary phase between the third and fourth day when cell density was about 4 x lo6 and 2.4 X lo6 cells/ml, respectively. The results are averages of two independent experiments.

SHORT

Hl” with the population doubling time and the extent of differentiation. Clearly, Hl” increased to comparable levels in induced (Fig. 3A) and control cells (Fig. 3B) although with different kinetics. Interestingly, Hl” accumulation does not correlate with differentiation but directly reflects the increase in population doubling time. MEL cell differentiation can be induced in vitro by a host of nonphysiological chemicals [29], including butyrate [30], although not all lines respond to the same inducers. The MEL clone JB3 that we have used does not differentiate when exposed to 1 mM butyrate (a nontoxic dose that slows cell growth to a rate comparable to that in 4 mM HMBA). Figure 2B shows that the Hl’ contents of cells exposed for 4 days to 1 mM sodium butyrate or to 4 mM HMBA were nearly identical. Since cells treated with HMBA did differentiate whereas those treated with butyrate did not, these results, together with those shown in Fig. 3, support the view that Hl” accumulation in differentiating MEL cells primarily reflects changes in the rate of cell division rather than the state of differentiation. DISCUSSION

Our results are in agreement with previous reports showing that the relative concentration of Hl” increases during MEL cell differentiation [15, 17, 31,321. However, from an analysis of cell growth in the presence and absence of chemical inducers, we interpret our observations as indicating that the higher Hl” expression is not necessarily linked to the state of differentiation but rather to the increased cell cycle length of induced and noninduced cells. In this regard, MEL cells behave like other nondifferentiating cells that show increased Hl” levels following inhibition of DNA replication or proliferation arrest (2-51. Although it remains possible that an increase in Hl” is needed for the establishment of the differentiated phenotype, given the low levels of Hl” of the clone used, it is clear that the Hl” levels that would be required are much lower than those previously reported for other MEL clones [l&17,31,32]. Relevant to this is the apparent absence of Hl” in splenic erythroblasts from anemic mice [33]. Although Hl’ occurs in different mammalian tissues and tumor cell lines, its highest content is found in tissues with a low rate of cell turnover ([6, 11,121; see also Fig. lA, lane 1). This could reflect the progressive replacement of replication-dependent Hl’s by Hl” resulting from histone turnover during tissue aging. Hl” may play no specific function in differentiation other than maintaining the supply of linker histone in the absence of the replication-dependent Hl’s. In other words, the role of Hl” may not reside in its particular structure but in the timing of its expression. The view that Hl” function may not be different from that of the other Hl

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subtypes is supported by the lack of an Hl” homologue in birds, since H5 is only expressed in erythroid cells ([34] and references therein). In birds, the role of Hl” appears to be fulfilled by certain members of the replication-dependent Hl family [35,36] which continue to be expressed in nonproliferating tissues by virtue of alternative processing of the mRNA [36]. The basis for the higher Hl” contents of differentiated tissues can be probably found in the fact that transcription of the Hl’ gene may be independent of the S phase. Although the molecular mechanisms that regulate the HI0 gene remain to be elucidated, reports indicating that the Hl” mRNA levels are, to some extent, independent of the S phase [ 14,371 are consistent with this possibility. As a consequence of this, Hl” could increase in relative concentration during the Gl and G2 phases of the cell cycle, when expression of the replication-dependent histone genes is down regulated, and be diluted out during S phase, when the latter are active [38]. Hence, alterations in the rate of cell growth, like those accompanying differentiation or mitogenic stimulation, would result in fluctuations of Hl” content, and the longer the life span of the cells in a tissue, the higher its content of Hl” would be, as could be the case for brain neurons and liver [6, 11, 121. It should be noted, however, that the absolute amount of Hl” of a given cell may depend not only on the relative level of its mRNA and those of other Hl subtypes (see [39]) but on the relative affinity of these histones for available chromatin binding sites (see [40]). This would explain why Hl” accumulates by a smaller factor than its mRNA during MEL cell differentiation [37] and the low levels of chromatin-bound Hl” of cells actively transcribing a transfected HI0 gene (A.R.-C., unpublished results). We thank Dr. W. Waithe for revising the English in the manuscript. J.B. thanks the Spanish Government for a postdoctoral fellowship (FPIIMEC). This work was supported in part by a grant from the Medical Research Council of Canada.

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