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(Received for publication, February 21, 1996)
From the ABSTRACT Effects of okadaic acid (OA), a protein
phosphatase inhibitor, on chromatin structure and phosphorylation of
histones were examined using HeLa and N18 cells. The chromatin
condensation in HeLa cells was mild and resemble prometaphase nuclei,
while the condensation in N18 cells was extensive and chromatin became
a compact body. H2A in HeLa cells was extensively and consistently
phosphorylated at the same site throughout the cell cycle, and H3 was
demonstrated to be phosphorylated at the mitosis-specific site
Ser10. In contrast, H1 phosphorylation was rapidly
decreased in most sites within 3 h. The reduction of H1 phosphorylation
was accompanied by a quantitative change in the set of H1
phosphopeptides. During the early phase of the OA treatment, H1
phosphorylation was transiently elevated in tandem, whereas H3
phosphorylation reached a maximum somewhat later. The results suggest
that mitosis-specific events (cdc2/H1 kinase activation, H1
superphosphorylation, mitosis-specific H3 phosphorylation and chromatin
condensation) induced by OA are sequentially associated. The changes
appear to reflect a molecular mechanism similar to that operating in
normal mitosis.
INTRODUCTION The control of cell cycle progression is one of the most basic
biological systems for which involvement of protein phosphorylation and
dephosphorylation has been shown (1, 2). These processes are controlled
by protein kinase and phosphatase activities. To understand the
mechanisms of chromosome kinetics in mitosis, it is thus crucial to
analyze the role of nuclear protein phosphorylations at the initiation
of chromatin condensation.
Histones constitute a major component of chromosomes and construct
nucleosomes as a basic chromatin element (3). During the cell cycle,
three kinds of histones (H1, H2A, and H3)1
are extensively phosphorylated in a histone-specific manner (4). During
the late G2 to M phase, the level of H1 phosphorylation
reaches a maximum (5, 6, 7, 8, 9) concomitantly with activation of
cdc2/H1 kinase (10, 11). Mitosis-specific H3 phosphorylation
occurs during M phase (4, 12) or premature chromosome condensation
(PCC) (13, 14, 15) at Ser10 (16, 17). The level of H2A
phosphorylation has been observed to be constant throughout the cell
cycle (4, 9).
Okadaic acid (OA), a specific inhibitor of serine/threonine protein
phosphatase (18, 19, 20) and a potential tumor promoter (21), is known to
affect mammalian cells and induce mitosis-like processes. The chemical
causes rounding of cells, PCC, spindle deconstruction and enhancement
of H1 kinase activity (22, 23, 24, 25). The chemical also induces apoptotic
cells (26, 27). These phenomena accompany an elevation of overall
cellular protein phosphorylations (28, 29). The inhibition of
phosphatase activity by OA is associated with increased phosphorylation
in which pp1 or pp2A phosphatases are involved. Thus it is possible to
investigate the relationship between chromatin condensation and histone
phosphorylation during cell proliferation using this agent (30).
In the present investigation, we examined critically the effects of
okadaic acid on chromatin condensation and histone phosphorylations in
both HeLa and N18 cells and found that OA induces H3 phosphorylation in
a mitosis-specific site concomitantly with chromatin condensation in
interphase cells.
MATERIALS AND METHODS OA was provided by Dr. S. Yamada of Nagoya
University or purchased from Wako Chemical Co. 6-Dimethylaminopurine
(6-DMAP) was purchased from Sigma Stock solution of OA was 1
mM dissolved in dimethyl sulfoxide.
HeLa (S-3) or N18 (mouse
neuroblastoma) cells were cultured in dishes (9-cm diameter, Falcon)
for 2 days. The cells were treated with or without chemicals for 30 min
to 2 h. For detailed observation of cell nuclei, cells were plated on
coverslips and treated with 0.1-1.0 µM of OA or 0.5-1.0
mM 6-DMAP. After washing two times with phosphate-buffered
saline solution, the cells were fixed with Carnoy's solution
(methanol:acetic acid, 3:1) for 30 min and stained with 50 µg/ml
Hoechst 33258 for 30 min, then washed twice with 90% ethanol and
observed by immunofluorescence microscopy (Nikon, Fluophot).
Cells were
grown in plastic bottles (Coster 3275). For the collection of mitotic
cells, logarithmically growing cells were treated with 0.02 µg/ml
colcemid for 16 h. Then, loosely attached cells were shaken off and
collected. For 32P labeling, cells (approximately 3.5 ×
107) grown either in dishes or in suspension culture were
resuspended in a phosphate-free medium (8) containing
[32P]orthophosphate (40 µCi/ml) with or without
chemicals. The cultures were incubated for 3 h or 30 min for pulse
labeling at 37 °C. Cells were partially lysed with a solution
containing 80 mM NaCl, 20 mM EDTA, and 1%
Triton X-100 (pH 7.6). Total histones were extracted twice from
chromatin with 0.4 N H2SO4 and the
proteins precipitated with 4 volumes of ethanol as described earlier
(14).
Samples containing 30 µg of histones were
resolved by electrophoresis on 30-cm-long 12% polyacrylamide gel
containing 7.5 M urea, 5% acetic acid, and 6
mM Triton X-100 (31). The proteins were run at 120 V for 48
h, and the gels were stained with 0.2% Amido Black and
autoradiographed as described previously (14). For the quantitation of
protein content, gels were subjected to scanning with a densitometer at
565 nm with a Digital Densitrol (type DMU-33C, Toyo Kogaku Co.). Each
protein band in the gel was cut out, and the radioactivity of
32P in the protein was estimated by Cerenkov counting.
The
procedures used for trypsin digestion and tryptic phosphopeptide
mapping of phosphorylated histones have been described previously (32).
Phosphorylated histones were resolved by SDS-polyacrylamide gel
electrophoresis (33). Individual histones was recovered
electrophoretically from gels. After trichloroacetic acid
precipitation, proteins were dissolved in 0.05 M sodium
bicarbonate and digested with 2% trypsin for 4 h at room temperature.
Resolution of the phosphopeptides in the first dimension was by
electrophoresis in buffer containing buthanol, acetic acid, water,
pyridine, 50:25:900:25 (pH 4.7) at 550 V for 50 min on cellulose TLC
plates (Merck, Art 5716) under Isoper (Esso Co.). After the plates were
dried, resolution in the second dimension was by ascending
chromatography in a solution containing n-butanol, acetic
acid, water, pyridine (36.6:11.4:45.3:56.7).
Cells
(4 × 106) were lysed partially with buffer A (10
mM Tris·HCl (pH 7.4), 3 mM MgCl2,
0.5 mM phenylmethylsulfonyl fluoride) containing 0.4%
Nonidet P-40. Nuclear pellet was washed once with buffer A without
Nonidet P-40. From the nuclear pellet, 0.4 M NaCl soluble
proteins were extracted. The extract was centrifuged at 100,000 ×
g for 30 min. The clarified extract was diluted with beads
buffer (34) and was reacted with p13suc1-Sepharose beads
(Oncogene Sci, Inc.) for 90 min. The beads were then washed three times
with beads buffer A. H1 kinase activity associated with the beads was
estimated in a reaction mixture (50 µl) containing 20 mM
Tris·HCl (pH 7.5), 1 mM EGTA, 1 mM
dithiothreitol, 10 mM MgCl2, 10 µg of HeLa
histone H1, 1 µM API (an inhibitor peptide for protein
kinase A, Sigma) and 20 µCi of [ RESULTS To investigate the
fine structure of HeLa nuclei treated with OA, cells on coverslips were
fixed and stained with DNA binding dyes (Fig. 1). Most
of the HeLa cells treated with OA at 0.1-1.0 µM detached
from the substratum. In the cells remaining attached, weak chromosome
condensation was observed in approximately 80% of nuclei (Fig.
1B), whereas untreated cells had round nuclei with a few
mitoses (Fig. 1A). The condensation in HeLa cells was mild
and resembled prometaphase nuclei. The extent of condensation varied,
suggesting that it depends on the cell stage at the time of
administration. Most mitotic nuclei were overcondensed with reduction
in size and rounding (Fig. 1B, indicated by an
arrowhead). In nuclei pretreated with colcemid for 16 h
prior to the addition of OA for 3 h, the number of prometaphase-like or
compact nuclei was remarkably increased (data not shown). However, most
nuclei of N18 cells were extensively condensed and showed round body
with no chromatin fibers (data not shown).
Since protein
phosphorylations are associated with chromosome condensation, histone
phosphorylations were examined. HeLa cells were labeled with
32P in the presence of OA or 6-DMAP, and the histones were
extracted and analyzed by acid-urea-Triton X-100 gel electrophoresis
(Fig. 2). Electrophoretic patterns of the proteins were
the same for both control and inhibitor-treated cells, except for
additional H2A bands caused by phosphorylation (Fig. 2A, lanes
2 and 3, H2A.1 and H2A.2). H2A and H1 are
extensively phosphorylated as observed in most growing cells (Fig.
2A, lane 7). In the OA-treated cells, H2A phosphorylation
was remarkably increased (Fig. 2A, lanes 8 and 9)
with two subtypes, H2A.1 and H2A.2, incorporating 2 and 2.5 times more
of 32P, respectively (Fig. 2A and Table
I). It was unexpected that the level of H1
phosphorylation was less than half of that in control cells. H3 was
rapidly phosphorylated in all three subtypes (H3.1, H3.2/H3.3) and
further increased with an increase in the OA concentration, whereas H2B
and H4 remained unphosphorylated. With the N18 cells (Fig.
2B), similar results to OA were obtained, although the
phosphorylation rate was lower than HeLa (Fig. 2B, lanes 8
and 9). The data therefore indicated that OA affected
histone phosphorylation in a histone-specific manner in the two
mammalian interphase cells. Thus, 1) H2A phosphorylation was increased,
2) H1 phosphorylation was extensively decreased, 3) H3 phosphorylation
was newly induced in interphase cells, and 4) H2B and H4 remained in an
unphosphorylated state as in the control cells, 6-DMAP inhibited both
H2A and H1 histone phosphorylations of HeLa cells (Fig. 2A, lanes
11 and 12). The chemical also suppressed the mitotic H3
phosphorylation induced by colcemid (Fig. 2A, lane 10). The
HeLa cells with colcemid contained approximately 48% mitotic cells.
The decrease of these phosphorylations corresponded to the treatment
dose (0.5 mM in lane 11 and 1.0 mM
in lane 12) and also to the extent of chromatin
decondensation (data not shown). A similar influence with histone
dephosphorylation by 6-DMAP was observed (Fig. 2B, lanes 11
and 12) in the N18 cells. Quantitative data for histone
phosphorylations in both HeLa and N18 cells in the presence of these
two chemicals are summarized in Table I.
Incorporation of 32P into individual histones (cpm/µg) in the
presence of OA or 6-DMAP with colcemid
Volume 271, Number 22,
Issue of May 31, 1996
pp. 13197-13201
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
INDUCTION OF MITOSIS-SPECIFIC H3 PHOSPHORYLATION AND CHROMATIN
CONDENSATION IN MAMMALIAN INTERPHASE CELLS*
§,
and
Aichi Cancer Center, Research Institute,
Laboratory of Cell Biology and 
Laboratory of Ultrastructure
Research, Chikusa-ku and the ¶ Nagoya University, Faculty of
Science, Institute of Molecular Biology, Nagoya 464, Japan
-32P]ATP (DuPont
NEN) with incubation at 30 °C for 15 min. The reaction was stopped
by adding 20% trichloroacetic acid on ice. H1 was precipitated and
washed once with acetone containing 0.3% HCl and twice with acetone.
Then, the proteins were run on SDS gels. Bands were detected by
autoradiography of dried gels. For quantitation, labeled bands were
excised, and radioactivity was determined by Cerenkov counting.
Fig. 1.
Chromatin condensation in the presence of
OA. HeLa (S-3) cells were cultured at 4.5 × 104/ml on
coverslips for 2 days, treated with 1 µM OA for 1 h,
washed with saline solution, and then fixed in methanol/acetic acid
(3:1) for 30 min. The cells were stained with 50 µg/ml Hoechest 33258
in 90% ethanol, washed twice with 90% ethanol, and dried. Observation
was under an immunofluorescence microscope. A, untreated
HeLa cells; B, cells treated with 1 µM OA for
1 h.
Fig. 2.
Incorporation of [32P]phosphate
into histones in cells in the presence of OA or DMAP. HeLa or N18
cells were labeled with [32P]phosphate (40 µCi/ml) for
3 h, and histones were extracted as described under ``Materials and
Methods.'' Forty µg (~3,500 cpm) aliquots of the proteins were run
on acid-urea-Triton gel electrophoresis at 120 V for 48 h. The gels
were stained with 0.2% Amido Black and then autoradiographed.
A, HeLa; B, N18. Lanes 1-3 show the
effects of OA on histone phosphorylations, and lanes 4-6,
the effects of 6-DMAP. Lane 1, control; lane 2,
0.5 µM OA; lane 3, 1 µM OA;
;lane 4, colcemid only; lane 5, colcemid with 0.5
mg/ml 6-DMAP; lane 6, colcemid with 1 mg/ml 6-DMAP.
Lanes 1-6, Amido Black stain and lanes 7-12,
32P autoradiography corresponding to lanes
1-6, respectively.
OA
6-DMAP with colcemid
0
µM
0.5 µM
1.0 µM
0
mM
1.0 mM
2.0 mM
HeLa
H2A.1
149.0
305.3 (2.04)
294.3 (1.98)
125.3
15.4 (0.12)
10.6 (0.08)
H2A.2
137.5
352.0 (2.56)
331.5 (2.41)
125.3
H3.1
6.4
200.3 (31.30)
245.2 (38.30)
38.1
H1
179.5
72.5 (0.40)
94.5 (0.53)
201.4
117.9 (0.59)
67.6 (0.33)
H3.2/.3
4.2
133.2 (31.71)
194.6 (46.30)
23.6
N18
H2A.1
81.5
100.5 (1.23)
104.6 (1.28)
80.0
49.1 (0.61)
26.0 (0.33)
H2A.2
69.3
98.6 (1.31)
90.5 (1.42)
70.4
52.5 (0.75)
26.5 (0.38)
H3.1
10.5
15.7 (1.5)
15.9 (2.8)
12.5
H1
90.6
31.6 (0.35)
19.9 (0.22)
127.1
98.8 (0.78)
37.2 (0.29)
H3.2/.3
8.3
10.6 (1.28)
25.6 (3.08)
11.0
ABSTRACTCritical examination of the effective dose of OA for induction of H3
phosphorylation and decreased H1 phosphorylation revealed clear
differences between N18 and HeLa cells (Fig. 3). The
minimum concentration of OA for induction of H3 phosphorylation in the
HeLa case was 0.2 µM (Fig. 3, lane 8), whereas
the induction in N18 cells at 0.2 µM was very low (Fig.
3, lane 3). The extent of H3 phosphorylation in N18 cells
rapidly increased with low concentrations of OA (0.1-0.5
µM) (Fig. 3, lanes 2-4), to a greater extent
than with colcemid (data not shown). H3 phosphorylation reached maximum
levels at 0.5 µM in HeLa cells (Fig. 3, lane
9), against 1 µM in N18 cells (Fig. 3, lane
10). In contrast, H1 phosphorylation decreased with an increasing
dose of OA in both cell types.
Fig. 3. Incorporation of [32P]Phosphate into H1 and H3 histones in cells treated with various concentrations of OA. 32P-Labeled histone preparation and analysis were conducted as described in the legend to Fig. 3. Histones (30 µg) were loaded on acid-urea-Triton gels and autoradiographed after electrophoresis. The figure shows only the H1 and H3 regions. Numbers 1-6 indicate HeLa histones for 0, 0.1, 0.2, 0.5, 1.0, and 1.5 µM of OA, respectively, and numbers 7-11, those from N18 cells treated with 0, 0.1, 0.2, 0.5, and 1.0 µM of OA, respectively.
Comparison of Histone Phosphopeptides between OA-treated Cells and M Cells
To analyze whether the changes of histone
phosphorylations by OA reflect any alteration in the number of
phosphorylation sites, [32P]phosphopeptide maps of the
three kinds of histones were prepared and compared with those of
control cells (Fig. 4). The phosphopeptide of H2A in the
OA-treated cells was single (Fig. 4b) and migrated to the same position
as with H2A in untreated cells (Fig. 4a). The H2A spot in
OA-treated cells was more extensively labeled than that in the control
H2A. The positions of individual H1 phosphopeptides with OA (Fig.
4d) were also similar to those in untreated cells (Fig.
4c), but the intensity of the individual phosphopeptide
differed considerably. The 32P radioactivity in most spots
was reduced and one spot demonstrated intensification (Fig.
4d, indicated by an arrow). The two major H3
phosphopeptides with OA (Fig. 4f) were the same as the two
mitotic H3 phosphopeptides (3-1 and 3-2 in Fig. 4e). The
amino acid sequences of the phosphopeptides, 3-1 and 3-2 were shown
previously to contain the same residue Ser(P)10; the
peptide 3-2 is a product of incomplete proteolysis (17). The results
indicate that OA does not induce an extra phosphorylation site in both
H2A and H1, but does induce H3 phosphorylation in interphase cells
exactly at the same site as in mitotic cells and some minor sites.
Fig. 4. Comparison of histone phosphopeptides in OA treated cells and mitotic cells. HeLa cells were treated with either OA (1 µM) for 3 h or colcemid (0.04%) for 16 h. For the final 3 h, cells were labeled with 32P in the presence of the chemical. H1 was extracted with 5% perchloric acid directly from nuclear pellet. H2A and H3 were obtained from total histones separated on SDS-polyacrylamide gels and electrophoretical elution. Fifty µg of H1 or 15 µg of H2A or H3 was digested with 2% trypsin. The minimum counts of radioactivity loaded on TLC plates were approximately 2,500 cpm for H1 and 1,500 cpm for H2A and H3. Resolution of the phosphopeptides on the TLC plates in the first dimension was carried out by electrophoresis, then by chromatography in the second dimension as described under ``Materials and Methods.'' a, H2A in untreated cells; b, H2A in OA-treated; c, H1 in untreated; d, H1 in OA-treated; e, H3 in mitotic cells; f, H3 in OA-treated. The arrow in d indicates a spot with increased relative intensity of 32P. 3-1 and 3-2 in e were identified as peptides containing Ser10 residue in an earlier study (17). Small black dots indicate the origin of the phosphopeptides.
Relationship between H1 Kinase Activity and H1 and H3 Phosphorylations in HeLa Cells in the Presence of OA
Since
OA-induced chromatin condensation in interphase cells, H1
superphosphorylation and mitosis-specific H3 phosphorylation were
investigated simultaneously (Fig. 5). The H1 and H3
phosphorylations in OA-treated cells were examined by pulse labeling
with 32P (Fig. 5a). The H1 kinase activity in
the 0.4 M NaCl-soluble nuclear fraction was examined
in vitro using HeLa histone H1 as a substrate (Fig.
5a). At the same time, H1 and H3 phosphorylations in
aliquots of OA-treated cells were examined by pulse labeling with
32P (Fig. 5b). During the first 30 min after the
OA treatment, the incorporation of 32P into H1 did not
essentially differ from that of untreated cells (Fig. 5, a
and b), and H3 phosphorylation was negligible. H1
phosphorylation was elevated and peaked after 1 h, and H3
phosphorylation reached a maximum level at around 1.5 h. Thereafter, H1
phosphorylation returned to the normal level by 2 h, whereas H3
phosphorylation was maintained at a relatively high level. The data
indicate that the kinetics of H3 phosphorylation differed from that of
H1 phosphorylation. Quantitative data on H1 kinase activity, H1
phosphorylation, and H3 phosphorylation are shown in Fig.
6.
Fig. 5. Time course of changes in cdc2/H1 kinase activity and H1 and H3 phosphorylations in the presence of OA. 32P-Labeled histones were prepared from OA-treated HeLa cells, and experiments were conducted in parallel with analysis of H1 kinase activity. After OA treatment, aliquots of cells were harvested at 30 min and 1, 2, and 3 h. The cells were lysed, 0.4 M NaCl soluble nuclear proteins were prepared, and H1 kinase activity was examined as described under ``Materials and Methods.'' Remaining cells were pulse-labeled for a 30-min interval with 32P, and histones were prepared. The 32P-labeled histones were run on acid-urea-triton gels. a, H1 kinase activity in the nuclear extract; b, the incorporation of 32P into histones H1 and H3.
Fig. 6. Relationship between H1 kinase activity and H1 and H3 Phosphorylations. The three items, H1 kinase activity in the nuclear extract (
) and the incorporation of 32P into
histones H1 (
) and H3 (
) in the presence of OA as shown in the
Fig. 5, were quantitated (cpm/µg) and depicted together.
DISCUSSION
The present study confirmed that OA causes interphase cells to demonstrate mitosis-like characteristics in terms of both morphological and biochemical parameters. Thus, 1) most OA-treated interphase cells became round in shape with condensed chromosomes, and 2) mitosis-specific histone H3 phosphorylation was induced, although the other cell cycle-dependent histone phosphorylations (H1 and H2A) were also influenced. Our data imply that OA inhibition of phosphatase results in accumulation of phosphorylated H3 proteins, so condensation of chromatin was induced without the completion of DNA synthesis. 6-DMAP, in contrast, decondenses chromatin and concomitantly dephosphorylates H3 even in cells pretreated with colcemid. The chemical is known to inhibit both MPF kinase activity and protein phosphorylations during the maturation of oocytes (35, 36). Therefore, 6-DMAP inactivates both H1 and H3 kinases and thereby dephosphorylates H1 and H3 phosphorylations, which are accumulated in the presence of colcemid, leading to chromatin decondensation. This result is consistent with that of Th'ng et al. (37), who used staurosporine, another protein kinase inhibitor, and found it induced both H1 and H3 dephosphorylations and chromatin decondensation in FM3A cells. This evidence suggests that chromatin condensation must be regulated by a protein kinase/protein phosphatase related mechanism.
OA-induced chromatin condensation of HeLa cells was relatively weak as compared with the case with N18 or other cells (22). Steinmann et al. (38) reported that chromatin in human and mouse cells is hard to condense by OA. The different extent of chromatin condensation among cell types may derive from different rates of H1 kinase induction. We found that induction of H1 kinase by OA was only two times enhanced in HeLa, against 10 times in baby hamster kidney cells (22). This might be due to a lower content of factors supporting H1 kinase activity, like cyclin (38).
It is known that the Ser/Thr-protein phosphatase inhibitor, OA, specifically inactivates both protein phosphatases, pp1 and pp2A. The pp2A is inhibited completely with 1-2 nM OA, whereas pp1 requires 1 µM (18, 20). At the low concentration of OA (2 nM) giving pp2A inhibition, no appreciable effect was observed in terms of H2A and H3 phosphorylations or chromatin structure. Therefore, the data indicate that H2A or H3 dephosphorylations during the cell cycle involve pp1 rather than pp2A.
The mechanism by which OA induces such a burst increase of H3 phosphorylation remains unclear, and the question of whether H3 can be phosphorylated in its natural state or whether the phosphorylation requires some structural change in chromatin still awaits an answer. The H3 phosphorylation itself was probably accelerated by OA inhibition of the relevant phosphatase rather than by H3 kinase activation, since Ser10 in nucleosomes associated with H1 is hardly phosphorylated in vitro (17, 39).
In the presence of OA, the H1 phosphorylation level decreased after 2-3 h in the present case. Paulson et al. (40) similarly found that mitotic H1 phosphorylation of HeLa cells was inhibited by OA using retardation of phosphorylated proteins in gel electrophoresis. The present data indicate that 1) dephosphorylation of H1 occurs not only in M cells but also in interphase cells and 2) the dephosphorylation occurs only in H1 but not in H2A or H3. One possible explanation for this might be that H1 kinase is inactivated by OA with longer treatment as reported by Schonthal and Feramisco (41). The possibility is supported by the evidence that H2A and H3 phosphorylations are not inhibited by protein kinases other than cdc2/H1 kinase. Alternatively, the protein phosphatase responsible for H1 phosphorylation in vivo is neither pp1, pp2A, or the other new protein phosphatase, as discussed by Paulson et al. (40). Although it has been reported that pp2A (42) or pp1 (43) is involved, the phosphatase responsible in mammalian cells has not been determined yet. The remaining phosphorylated H1 demonstrated that the dephosphorylation of a specific peptide was very slow or uneffected by OA. This indicates that the efficiency of dephosphorylation by a protein phosphatase may depend on the primary structure of the phosphorylation site (44).
The present study revealed that H3 phosphorylation associated with OA occurs at the mitosis-specific site (Ser10). H3 phosphorylation is the most closely associated with chromatin condensation among the three phosphorylatable histones during the cell cycle. Extensive H3 phosphorylation has been observed thus far with various kinds of chromatin condensation; 1) normal mitosis from prophase to anaphase (4), 2) PCC in a hybrid cells between S and M phase cells (13), 3) PCC in tsBN2 at the nonpermissive temperature (14, 15), and 4) PCC in the presence of protein phosphatase inhibitor, OA (present study). In most cases, there is an accompanying high level or transient elevation of H1 phosphorylation. It was earlier proposed that a high level of mitotic H1 phosphorylation may facilitate mitotic H3 phosphorylation (17, 39). It is possible that this site of H3 could become exposed by the transient elevation of H1 phosphorylation and thereby become accessible to H3 kinase. Subsequent to this step, phosphorylated H1 may not be directly involved in chromatin condensation until H1 phosphatase removes the phosphate. This notion may explain the recent controversy regarding the role of H1 in chromatin condensation in that it is not essential in the amphibian embryonic system in vitro (45, 46), whereas it is in growing organisms (47). On the other hand, it is known that OA phosphorylated on 80-kDa proteins binding at AT-rich and on nuclear matrix-associated 70-kDa proteins (48). The effect of these phosphorylated non-histone proteins is not rule out for the chromatin condensation.
The H1 kinase activated by OA is known to be Cdc2-type protein kinase associated with cyclin B, which is same as in mitotic cells (22). The present evidence that transient H1 and mitotic H3 phosphorylations occur in the presence of OA indicates that chromatin condensation with OA in interphase cells is induced by a biochemical mechanism similar to that operating in normal mitosis. The causal relationship between chromatin condensation and H3 phosphorylation now requires elucidation, as well as determination of H3 kinase(s) actions.
FOOTNOTES
§ To whom corresponding should be addressed: Aichi Cancer Center, Research Institute, Laboratory of Cell Biology, Chikusa-ku, Nagoya 464, Japan. Tel.: 052-762-6111 (ext. 8821); Fax: 052-763-5233.
1 The abbreviations used are: H, histone; OA, okadaic acid; 6-DMAP, 6-dimethylaminopurine; PCC, premature chromosome condensation; pp1/pp2A, protein phosphatase 1 and 2A.
Acknowledgment
We gratefully acknowledge the guidance and constant encouragement of Dr. T. Okazaki.
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