Download Relationship between the timing of DNA replication and the

Relationship between the timing of DNA replication and the developmental
competence in Acanthamoeba castellanii
HELGA JANTZEN, INGRID SCHULZE
Zoologisches Institut II, Universttcit Heidelberg, Im Neuenheimer Feld 230, 6900 Heidelberg, West Germany
and MICHAEL STOHR
Institut fur Experimentelle Pathologie, Deutsches Krebsforsclwngszentrum, Im Neuenheimer Feld 280, 6900 Heidelberg, West Gennanv
Summary
In Acanthamoeba, two different cell types are
known. Trophozoites are generated in the mitotic
division cycle, whereas cells committed at late G2
phase of the cell cycle develop into cysts in response
to starvation. In this paper we study the role of
timing of DNA replication in regulating development. The investigation was performed with cultures growing in a non-defined medium (ND cells)
that show a high encystation competence and with
cultures that have been growing in a chemically
defined medium (D cells) for several years and
show a low encystation competence. Bivariate
DNA/BrdUrd distributions show that ND cells progress through a cycle in which the short replication
phase occurs immediately and exclusively after
prior completion of mitosis. These cells arrest at
late G2 phase of the cell cycle during the stationary
stage. In D cells, DNA replication and mitosis seem
to be uncoupled, since replication takes place before as well as after mitosis. These cells arrest
within their replication phase during the stationary
stage. These findings indicate that D cells do not
progress into late G2 phase of the cell cycle and
hence do not have the competence for commitment.
The alternate timing of DNA replication and the
low encystation competence of D cells can be
reversed by cultivation of these cells in ND medium. Synchronization experiments reveal that late
G2 phase ND cells exhibit a low capacity for BrdUrd
incorporation and growth after transfer into D
medium, whereas ND cells of earlier phases of the
cell cycle show premitotic incorporation of BrdUrd
into nuclear DNA and growth. These findings
suggest on the one hand that premitotic DNA synthesis is a prerequisite for growth of cells in D
medium, and that there is a dependence of the
induction of premitotic DNA synthesis on the cell
cycle, and on the other hand that a reciprocal
relationship exists between the capacity of premitotic DNA synthesis and commitment to differentiation.
Introduction
and cytokinesis. When cells of the growth stage are
transferred into non-nutrient medium, only 7 % of cells
encyst, whereas SO to 70% of cells of a stationary stage
culture develop into cysts. Synchronization of cells by
release from the stationary stage reveals that encystation
is initiated at a particular time in late G 2 phase. This
result suggests that in contrast to growing cells, the high
encystation competence of stationary stage cells is due to
arrest of cells at this particular point in G 2 phase. These
findings were obtained with cells cultivated in a nondefined medium containing proteose peptone and yeast
extract (ND cells). In contrast, cells that have been
adapted to a chemically defined medium containing 11
amino acids (D cells) exhibit a low encystation com-
The unicellular eukaryote Acanthamoeba castellanii has
the option of two pathways of cell-type generation.
Trophozoites are generated in the mitotic division cycle,
whereas cysts are generated in the differentiation pathway. The relationship between particular cell cycle
phases and differentiation into cysts has been investigated
recently (Stohr et al. 1987) and may be summarized as
follows. In trophozoites flow fluorometric measurements
of nuclear DNA content and determination of bivariate
DNA/BrdUrd distribution indicate a lack of Gi phase
nuclei and a cell cycle start with a short S phase (about
0-5 h) followed by a long G 2 phase (about 7h), mitosis
Journal of Cell Science 91, 389-399 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Key words: Acanthamoeba, cell cycle, DNA replication,
development, bivariate DNA/BrdUrd distribution.
389
petence. On the basis of the results obtained with ND
cells, it was suggested that the low encystation competence of D cells is due to a loss of accumulation of
stationary stage D cells at the particular position of G 2
phase. This suggestion is supported by the finding that
the nuclear DNA content of stationary stage D cells is
about 15 % lower than in G 2 phase cells. Furthermore,
growing D cells indicate altered nuclear states, since in
10-20% of cells, an increase of up to 40% of the G 2
phase DNA content can be observed.
In an effort to study a possible relationship between the
timing of DNA replication and developmental competence, this study examines more closely the dynamics
of nuclear DNA synthesis in Acanthamoeba cultivated in
a chemically defined medium. The results show that in D
cells DNA synthesis takes place not only immediately
after completion of mitosis as in ND cells, but also before
mitosis. The occurrence of premature DNA replication is
related to the inconstancy of nuclear DNA content as well
as to the low competence of development in these cells.
Materials and methods
Conditions of growth
Cultivation of Acanthamoeba castellaiiii (Neff strain) in a nondefined yeast extract/proteose peptone medium (ND cells) or
in a chemically defined medium (D cells), and encystation in
non-nutrient medium, were carried out as described (Jantzen,
1974; Jantzen & Schulze, 1987). In order to obtain synchronously growing ND cultures, standard stationary stage cells were
taken 3 days after the termination of growth and diluted out into
fresh non-defined nutrient medium (Stohr et al. 1987).
Simultaneous flow cytometric analysis of total DNA
content and incorporated 5-bromodeoxyuridine
(BrdUrd) into nuclear DNA
Labelling of cells by concomitant addition of BrdUrd and
fluorodeoxyuridine (FdUrd), preparation of cells for flow
cytometric analysis by staining of fixed cells with fluorescein
(FITC)-conjugated anti-BrdUrd and propidium iodide (PI),
and the flow cytometric measurements of bivariate DNA/
BrdUrd distribution were carried out according to Dolbeare et
al. (1983) as described (Stohr et al. 1987). Analysis of stained
cells by fluorescence microscopy revealed that the FITC label
was located exclusively and the PI label mainly within the
nuclei. Cells not pulsed with BrdUrd/FdUrd, but stained with
anti-BrdUrd/PI showed no anti-BrdUrd staining.
Restriction fragment analysis of nuclear and cellular
DNA
Nuclei were prepared by the Triton lysis method as described
by Stohr et al. (1987). For conventional DNA preparation,
SxlO 7 nuclei were digested with 100 fig proteinase K in l m l
45 mM-EDTA, 80mM-NaCl, 0-5% SDS (pH7-8) for 2h at
37°C. After extraction with phenol and chloroform/isoamyl
alcohol and DNA precipitation, the DNA was incubated in the
presence
of
deoxyribonuclease-free
ribonuclease
A
(100 ^gml" 1 ) in 80mM-NaCl, 5 mM-EDTA (pH7-5) for O-Sh
at 37°C, and thereafter in the presence of proteinase K
(50,ugml~ ) for 0-5 h. Following extraction with phenol/
chloroform/isoamyl alcohol, the DNA was precipitated in the
presence of 02M-sodium acetate with 2 5 vol. ethanol.
DNA was also prepared by embedding the cells or nuclei in
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H. Jantzen et al.
agarose blocks and then incubating the blocks with proteinase K
(Schwarz & Cantor, 1984). Cells or nuclei were suspended in
PBS (015M-NaCl, 005M-Na 3 PO 4 (pH7-2)) and mixed with
an equal volume of a 1 % solution of low melting agarose in PBS
at 42°C. The mixture was immediately poured into a slot former
so that an 80^1 agarose block contained 2X10 6 cells or nuclei.
Blocks were incubated for 48 h at 55 °C in 1% iV-lauryl
sarcosine, 0-5M-EDTA (pH8) with proteinase K ( 2 m g m r ' ) ,
and then stored in 0-5 M-EDTA (pH 8). For enzyme digestion,
blocks were rinsed in T E (10mM-Tns-HCl, 1 mM-EDTA
(pH8)) and incubated twice in TE containing 0-04 mgml" 1
phenylmethylsulphonyl fluoride for 30min at 55°C. For cleavage of DNA with restriction endonucleases, blocks were washed
in TE and a 40,ul block was incubated with 17 units of enzyme
for 4 h in 200 jA restriction enzyme buffer as recommended by
the suppliers. A 20 /.A block containing 0-5 xlO 6 cells or nuclei
was placed into a mixture of 0'5 X TBE gel running buffer
(45mM-Tris base, 45mM-boric acid, 1-25 mM-EDTA) and
0-1 vol. of 10X dye solution (50% (w/v) Ficoll, 0-2% Bromphenol Blue, 400 mM-EDTA) and then loaded into a slot of a
0-3% agarose gel. Electrophoresis was performed at 25 V for
14 h and 40 V for 8h. Following electrophoresis, gels were
stained for 30min in 0-2^gml~' ethidium bromide and photographed. DNA samples prepared either by the lysis or by the
agarose block method contained about 0-5 fig DNA (the nuclear
DNA content of Acanthamoeba is 1-2 pg cell" 1 ).
Results
Comparison of the timing of DNA synthesis in D and
ND cells
Growing D and ND cells were labelled with BrdUrd,
fixed and subsequently stained with FITC-conjugated
anti-BrdUrd to show nuclear DNA synthesis and with PI
to show nuclear DNA content. Analysis by flow cytometry yielded bivariate DNA/BrdUrd distributions.
When cells are labelled for 0-5 h, contour plots indicate a
small FITC/PI-labelled cohort that exhibits a lower
DNA content than the major FITC-negative, Pi-positive
cell population (Fig. 1). From similar results it has been
concluded that the S phase in both D and ND cells takes
place immediately and exclusively after completion of
mitosis (Stohr et al. 1987). However, BrdUrd/FdUrd
labelling of growing cells for extended time periods
reveals obvious differences between bivariate DNA/
Fig. 1. DNA synthesis in exponentially growing cells, shown
by simultaneous flow cytometric analysis of total DNA
content and incorporated BrdUrd. A,B. Exponential ND
cells (A) and D cells (B) were grown in the presence of
BrdUrd/FdUrd for several hours. At the indicated times cells
were fixed and stained with FITC-conjugated anti-BrdUrd
and PI. The increase in the relative cell number during the
labelling period is indicated in parenthesis. In two examples
individual cell populations are indicated by arrowheads.
Arrowhead 1. First FITC-positive cohort containing
postmitotically replicating cells. Arrowhead 2, second FITCpositive cohort containing cells in which DNA replication has
taken place. Arrowhead 3, third FITC-positive cohort
containing premitotically replicating cells. Owing to the
stickiness of cells in some cases cell doublets are also to be
seen. As examined by fluorescence microscopy, labelled cells
contain one FITC-positive nucleus. Abscissa: content of
DNA in arbitrary fluorescence units (PI). Ordinate: content
of incorporated BrdUrd in arbitrary fluorescence units
(FITC).
T
64 -
m
DNA content
Acanthamoeba, DNA replication and development
391
u
8
•s
•a
ffl
DNA content
Fig. 2. Bivariate DNA/BrdUrd distribution in labelled log stage D cells transferred into arginine-deficient medium.
A,B. Growing D cells were labelled with BrdUrd/FdUrd for 4 5 h. At this time 27% of nuclei were FITC-positive. These cells
were then transferred into arginine-deficient, BrdUrd/FdUrd-containing medium and examined after 1 h (A) and 18 h (B). At
18 h 33 % of nuclei were FITC-positive.
BrdUrd distributions in D and ND cells. These differences may be described qualitatively as follows: First, the
rate of increase of FITC-labelled cohort per labelling
time is lower in D cells than in ND cells. Second, in D
cells, especially after a prolonged period of BrdUrd/
FdUrd labelling, FITC fluorescence is found in a first
small cohort, in a second major cohort and in a third
smaller population. In ND cells, however, the third
FITC-positive cohort seems to be either absent or very
small. The first small FITC-positive cohort represents
postmitotic, DNA-synthesizing cells and the second
represents cells that have finished their S phase. The
third cohort, clearly seen in D cells, represents premitotic
cells in which DNA synthesis is, nevertheless, taking
place. This interpretation of bivariate DNA/BrdUrd
distributions is supported by the following observations.
Out of the three FITC-positive cohorts in D cells and the
two in ND cells, only the second FITC-positive cohort
increases during the labelling period, whereas the FITCnegative cohort concomitantly decreases. Hence, this
cohort should represent cells that have finished their S
phase. In this case, the distribution of the second FITCpositive cohort (representing G2 phase cells that have
progressed through the S phase) should coincide with the
distribution of the FITC-negative cohort (also representing G2 phase cells that, however, have not entered
S phase). This is obviously not the case in D cells.
However, when DNA synthesis in growing BrdUrdlabelled D cells is inhibited by deficiency of essential
amino acids, the distributions of the second FITCpositive and the FITC-negative cohort coincide (Fig. 2).
Thus, since the first FITC-positive cohort exhibits a
somewhat lower DNA content than the second, this first
cohort represents post-mitotic S phase cells. From the
observation that the third FITC-positive cohort, clearly
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observed in D cells, exhibits a higher DNA content than
the second FITC-positive cohort, it follows that this
cohort represents premitotic, DNA replicating cells.
Thus, these results support the recent finding that ND
cells progress through a cell cycle in which DNA
replication takes place immediately and exclusively after
completion of mitosis. In D cells, however, DNA synthesis takes place not only after nuclear division but also
before division.
Despite the occurrence of premitotic DNA synthesis in
D cells, the following results indicate that DNA synthesis
in these cells also correlates with cell proliferation. When
a cell population is in balanced exponential growth, the
slope of the regression line obtained by plotting the
percentage of BrdUrd-labelled nuclei (labelling index)
against labelling time roughly indicates the rate of entry
of percentage of cells into S phase perh. The duration of
the S phase may be estimated from the negative intercept
of the line with the horizontal axis, and the percentage of
cells of a growing culture being in S phase by extrapolation of the line to zero time (Sasaki et al. 1987). Fig. 3
shows that in both cultures, the labelling index increases
linearly with the labelling time up to an increase in the
relative cell number of about 1-7-fold. The duration of
the phase of DNA synthesis in D cells seems to be slightly
higher (0-55 ± 0-05 h, 3 experiments) than in ND cells
(0-3 ± 0-05 h, 2 experiments). Relative to ND cells, the
rate of entry of D cells into S phase is 1-7-fold lower,
corresponding approximately with the slower growth rate
of these cells. However, Fig. 3 also indicates an obvious
difference between the growth characteristics of D and
ND cells. Unlike ND cells, cytokinesis in D cells seems
to take place in several crises of short duration.
On the basis of the finding of a correlation between
BrdUrd incorporation and cell growth, the following
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Fig. 3. The relationship between labelling index, labelling time and growth rate in ND and D cultures. A,B- Growing ND
cells (A) and D cells (B) were labelled with BrdUrd/FdUrd. ( •
• ) The percentage of FITC-positive nuclei (labelling
index) was examined by fluorescence microscopy and plotted against labelling time. The regression lines were obtained by the
least-squares method. (O
O) The cell number in the labelled cultures was measured with a Coulter counter. (X
X)
Cell number of control cultures that were not labelled with BrdUrd/FdUrd.
suggestion may be made with regard to the appearance of
premitotic DNA synthesis associated with an atypical
nuclear DNA content in D cells. If premitotic DNA
synthesis indicates endoreduplication of part of the
genome twice instead of once or a severalfold amplification of particular DNA sequences, then total nuclear
DNA content should increase with increasing generations, unless either DNA is degraded in subsequent
cycles or excess DNA synthesis before mitosis consists
only of duplication of a section of the genome, and is
followed by non-replication of that section in the subsequent generation.
A rough estimation of DNA amplification can be made
by restriction fragment analysis of DNA. The Acanthamoeba genome exhibits a relatively low genetic complexity of 3X107 base-pairs (bp) (Jantzen, 1973) and
therefore a 10-fold increase in copy number of particular
DNA sequences may be detected by direct visualization
of ethidium bromide-stained gels of separated restriction
fragments (Coderre et al. 1983). By conventional preparation of DNA from D cell nuclei, we were unable to
obtain high molecular weight DNA, whereas with ND
nuclei a considerably lower degree of degradation occurs.
However, by embedding the cells or nuclei in agarose
blocks and subsequently digesting protein with high
concentrations of proteinase K, non-degraded DNA can
be obtained (Fig. 4). Fig. 4 shows restriction fragment
analysis of DNA prepared from cells and nuclei. In the
restriction pattern of total cellular DNA, several distinct
fragments are visible, however, these are due to the
presence of mitochondrial DNA. The characteristic restriction fragment pattern of the Acanthamoeba Neff
strain mitochondrial DNA and the genome size of
4 x l 0 4 b p that is obtained are in agreement with recent
results (Bohnert & Gordon, 1980; Bogler et al. 1983).
Since, in relation to the nuclear DNA complexity of
3 x l 0 7 b p (Jantzen, 1973), the mitochondrial DNA complexity of 4 x l 0 4 b p is 750-fold lower and the DNA
content about fivefold lower (Byers, 1979), a copy
number of about 150 of mitochondrial DNA per cell may
be assumed. Thus, if the excess nuclear DNA observed
in about 20 % of growing D cells is due to amplification of
particular DNA sequences, a copy number of about 100
(i.e. 20-fold amplification per total cells) should be
detectable. The restriction pattern of nuclear DNA
shows that this is not the case (Fig. 4). These results,
however, do not exclude the occurrence of DNA amplification of low frequency. The alternative interpretation is
that the cell cycle is altered in such a way that DNA
replication is begun before mitosis and completed after
mitosis. Several observations could support this suggestion: (1) postmitotic cells of D cultures (the first FITCpositive cohort in DNA/BrdUrd distributions) already
exhibit an enhanced F I T C / P I fluorescence (Fig. 1); (2)
it has been shown (Pritchard & Lark, 1964) that if a
culture is deprived of required amino acids, cells in the
process of a cycle of DNA replication complete that cycle
but are unable to initiate a new one. This also seems to be
the case in D cells. Fig. 3 shows that about 5 % of cells of
a growing D cell culture are engaged in DNA synthesis.
Acanthamoeba, DNA replication and development
393
12 3 4 a C
1 2 3 4 5 6 7 8 9 101112131415163 C
Fig. 4. Restriction fragment analysis of total cellular DNA
and nuclear DNA in ND and D cultures. A. Nuclear DNA
was prepared conventionally by incubation of nuclear lysates
in the presence of proteinase K and separated by 0-3 %
agarose gel electrophoresis. Lane 1, log stage ND; 2, log
stage D; 3, stationary stage ND; 4, stationary stage D.
B. Total cellular or nuclear DNA was prepared by
embedding of log stage cells or nuclei into low-melting
agarose and digestion with proteinase K. Lane 1, ND cells;
2, D cells; 3, ND nuclei; 4, D nuclei. C. Cleavage of cellular
and nuclear DNA was performed by incubating DNAcontaining agarose blocks in the presence of several restriction
endonucleases. Lanes 1-8, 9-16, restriction fragments of
cellular and nuclear DNA, respectively. Hindlll, ND
(lanes 1, 9) and D (2, 10); Xbal, ND (3, 11) and D (4, 12);
EcoRI, ND (5, 13) and D (6, 14); BamHl, ND (7, IS) and
D (8, 16). Size standards were obtained by using nondigested ADNA (lane a), a Sail digest (lane b) and a Hindlll
digest of ADNA (lane c).
Accordingly, during an 18 h BrdUrd/FdUrd labelling
period of an arginine-deficient log stage culture, 4-8 % of
nuclei become FITC-positive. Arginine is one of the five
essential amino acids in Acanthamoeba (Dolphin, 1976).
Thus, in response to amino acid deficiency, the bivariate
DNA/BrdUrd distribution should lack the first FITCpositive cohort (postmitotic) and the third cohort (premitotic), whereas the percentage of cells of the second
FITC-positive cohort (G2 phase cells that have finished
their DNA replication) should be increased by about
5%. In fact, this is the observed behaviour (Fig. 2). (3)
Recent experiments revealed that stationary stage ND
cells are arrested at late G2 phase of the cell cycle (Stohr
et al. 1987). Thus, if D cells are also stalled at a late cell
cycle phase and replication takes place both before and
after mitosis, stationary stage D cells should be stalled
within their replication phase. To address this question,
the distribution of bivariate DNA/BrdUrd and growth
were examined in stationary stage D cells transferred into
fresh D medium. Figs 5F and 6B show that during the
lag of cytokinesis, the FITC and the PI labels in the cells
increase slightly, indicating the arrest of cells within the
replication phase.
Taken together, ND cells progress through a cell cycle
in which a short replication phase (about 4 % of the cell
cycle) occurs immediately and exclusively after completion of mitosis. In D cells the duration of the
394
H. Jantzen et al.
replication phase is similar; however, DNA replication is
observed in postmitotic as well as in premitotic cells,
indicating an alteration in the timing of DNA replication.
Relationship between the timing of DNA replication and
the competence of encystation
Recent results indicate that stationary stage ND cells are
committed to encystation whereas stationary stage D cells
are not (Stohr et al. 1987). In an effort to test the
hypothesis that the incapacity of D cells to become
committed to development is due to the altered timing of
DNA replication, D cells were cultivated in ND medium. The occurrence of premitotic DNA synthesis in
log stage cells was related to the encystation competence
of stationary stage cells. Fig. 7B shows that immediately
after transfer of log stage D cells into ND medium,
premitotic DNA synthesis still takes place. When these
cells reach the stationary stage for the first time, encystation can be initiated in only 15-30 % of cells. However,
after further cultivation of D cells in ND medium,
distributions of bivariate DNA/BrdUrd are indistinguishable from those obtained with ND cells (Fig. 1),
and stationary stage cells show a high level of encystation.
Thus, the timing of DNA replication depends on the
growth condition of cells and the encystation competence
of cells depends on the timing of DNA replication.
As shown in the preceding section, the consequence of
an altered timing of DNA replication is that stationary
stage D cells are arrested within their replication phase,
whereas stationary stage ND cells are stalled at late G2.
Since stationary stage D cells show a low competence of
encystation, whereas stationary stage ND cells show a
high competence, it may be suggested that these nuclear
states are antagonistically mutually exclusive with regard
to commitment. It is not, however, the status of stalled
replication that is antagonistic to the status of commitment, since log stage G2 phase ND cells are also not
committed for encystation. Thus, it may be suggested
that commitment is inhibited in cells that express irregular (i.e. non-postmitotic) replication (D cells) or have the
capacity for expression. In an effort to test this possibility, the capacity for non-regular DNA synthesis in log
stage ND cells that do not encyst was compared with that
in stationary stage ND cells that do encyst. When log
stage ND cells are pelleted by low-speed centrifugation
(50^, 1-5 min, rav 12cm) and resuspended in fresh ND
medium, a small percentage of cells divide but the
majority of these cells do not incorporate BrdUrd and do
not divide for about 3 h. Thereafter, coordinated DNA
synthesis and cytokinesis takes place (Fig. 5A). A control
culture that was centrifuged in the same manner and
resuspended in its supernatant did not show this lag in
growth, indicating that cell growth is not perturbed by
this mode of centrifugation. In contrast, when pelleted
log stage ND cells are resuspended in D medium, DNA
synthesis is initiated immediately, and during the 3 h lag
of cytokinesis 15 % of nuclei become FITC-positive
(Fig. 5B). Since these cells contain only one FITCpositive nucleus, this indicates the induction of nonpostmitotic DNA synthesis. The distribution of bivariate
DNA/BrdUrd shows that incorporation of BrdUrd was
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found both in cells that had a PI fluorescence similar to
BrdUrd-negative cells and in cells that had a higher PI
fluorescence than BrdUrd-negative cells. This indicates
the occurrence of concomitant DNA degradation
(Fig. 7A). The induction of premitotic DNA synthesis is
inhibited by the absence of any of the five essential amino
acids (Fig. 5C). When growing D cells are transferred
into fresh medium, only a short lag of DNA synthesis and
cytokinesis occurs (Fig. 5D). However, in stationary
stage ND cells, neither BrdUrd incorporation nor cell
growth can be induced by transfer of cells into D medium
(Figs 5E and 6A). At 24 h after transfer, about 40% of
cells die, and the remaining cells seem to have encysted
incompletely.
The low capacity of stationary stage ND cells for
initiation of premitotic DNA synthesis and proliferation
after transfer into D medium may be due either to the
stationary stage of cells or related to the specific cell cycle
position at which cells are stalled. Recent results show
that transfer of stationary stage ND cells into fresh ND
medium leads to synchronous growth and concomitant
Fig. 5. Cell growth and DNA synthesis in
ND and D cells after transfer into either
fresh ND or D medium. Cells were pelleted
by low-speed centrifugation (50g, 1-Smin,
/•av 12 cm) and resuspended in fresh medium
containing BrdUrd/FdUrd. The percentage
of FITC-positive nuclei was determined by
fluorescence microscopy. A. Log stage ND
cells in ND medium. B. Log stage ND cells
in D medium. C. Log stage ND cells in
leucine-deficient D medium for 4-5 h, and
after re-addition of leucine. D. Log stage D
cells in D medium. E. Stationary stage ND
cells in D medium. F. Stationary stage D
cells in D medium. ( 0
• ) Percentage of
FITC-positive nuclei. (O
O) Relative
cell number. (X
X) Relative cell
number in a control culture, centrifuged
likewise, however, suspended in its own
supernatant.
DNA synthesis after a lag of 4h (Stohr et al. 1987).
Thus, the low capacity of stationary stage ND cells for
growth in D medium is not due to a general reduction in
the capacity for growth and regular postmitotic DNA
synthesis. Results, to be shown elsewhere, indicate a
decrease in the rate of pinocytosis (examined by the
transport of FITC-conjugated dextran into cells) in
stationary stage cells in comparison to log stage cells.
Since postmitotic and premitotic DNA synthesis in D
cells as well as in log stage ND cells transferred into D
medium is dependent on amino acid availability (Figs 2
and 5C), the rate of FITC-dextran transport was
measured in log stage and stationary stage ND cells
transferred into fresh D medium. However, no differences were found. Since pinocytosis seems to be the
major mechanism for uptake of dissolved substances
(Byers, 1979), this indicates that the low capacity of
stationary stage ND cells for initiation of premitotic
DNA synthesis and growth in D medium is not caused by
nutrient limitation. Thus, the reduced capacity of
stationary stage ND cells for growth in D medium may be
Acanthamoeba, DNA replication and development
395
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Fig. 7. DNA synthesis in log stage ND cells after transfer into D medium and in D cells after transfer into ND medium.
A,B. The incorporation of BrdUrd was studied in log stage ND cells after transfer into D medium (A) and in log stage D cells
after transfer into N D medium (B). After the indicated periods of cultivation cells were examined.
related to the particular cell cycle position at which these
cells are arrested. To follow the growth of ND cells in D
medium during the cell cycle, cells were synchronized by
release from the stationary stage and transferred into D
medium at various times. Fig. 8 shows that the proliferation property of late G2 phase cells is low in comparison
with cells of other cell cycle phases.
Taken together, the results reveal a relationship between the timing of DNA replication and developmental
competence of cells. The only cells that become arrested
at late G2 stage and are committed to development are
those cells in which DNA replication is exclusively
postmitotic. Since in committed cells premitotic DNA
synthesis is not inducible, it may be suggested that a
reduction in the capacity for non-regular DNA synthesis
is a prerequisite for development.
Discussion
The cell cycle timing of ND cells is characterized by a
short S phase (about 0-3 h), which takes place immediately and exclusively after completion of mitosis, and a
long G2 phase (about 7h). Accordingly, as measured by
flow cytometry, a unimodal distribution of nuclear DNA
content has been obtained. In growing D cells, however,
about 20% of nuclei can show an increase in DNA
content of up to 40 % (Stohr et al. 1987). The appearance
of a subset of cells with increased DNA content per cell
corresponds with the finding that DNA synthesis in D
Fig. 6. DNA synthesis in stationary stage ND and D cells
after transfer into fresh D medium. A,B. Stationary stage
ND cells (A) and D cells (B) were transferred into fresh D
medium containing BrdUrd/FdUrd. At the indicated times
cells were prepared for flow cytometric analysis (the
corresponding relative cell numbers are put in brackets).
2-5
., J
2-0
1-5
10
o
O—O
0
O
2
>
O-O-"
4
6
8
Time (h)
10
12
Fig. 8. The variation in the capacity of growth in D medium
of synchronously growing ND cell cultures. Synchronization
of cell growth was performed by release of stationary stage
ND cells into fresh ND medium. At the indicated times, cells
were transferred into D medium. The increase of cell number
was determined after 24 h. (O
O) Cell number of
synchronously growing cells in ND medium. (X
X) Cell
number of synchronous ND cells cultivated in D medium.
(®) Cell number of exponentially growing ND cells 24 h after
transfer into D medium.
cells takes place before as well as immediately after
nuclear division. The second difference between the
nuclear states of stationary stage D and ND cells also
seems to be related to the alternate timing of DNA
replication in D cells. ND cells that replicate exclusively
and early during the cell cycle, are stalled at late G2
phase, whereas D cells, which also replicate at a late
phase of the cell cycle, are arrested within their replication phase.
Acanthamoeba, DNA replication and development
397
The suggestion that premitotic incorporation of
BrdUrd in D cells indicates partial premature S phase
DNA replication seem to be supported by several
findings. (1) Distributions of bivariate DNA/BrdUrd
indicate that premitotic incorporation of BrdUrd is
associated with an increase in DNA content and that, in
comparison to ND cells, postmitotic D cells already
exhibit an elevated F I T C / P I content (Fig. 1). (2) After
inhibition of initiation of DNA synthesis by essential
amino acid deficiency, neither postmitotically nor premitotically DNA synthesizing cell populations are present
in bivariate DNA/BrdUrd distributions. The percentage
of FITC-positive G2 phase cells increases to equal the
proportion of DNA synthesizing cells seen before the
onset of amino acid deficiency (Fig. 2). (3) The results
indicate that premitotically synthesized DNA does not
consist of a high copy number of particular DNA
sequences (Fig. 4). However, DNA amplification of low
frequency cannot be excluded. On the other hand, the
finding that nuclease activity in D nuclei is higher than in
ND nuclei (Fig. 4), and that transfer of log stage ND
cells into D medium induces premitotic DNA synthesis
and presumably also DNA degradation (Fig. 7A), may
indicate the possibility that at least part of the excess
DNA is degraded.
In another amoeba, Amoeba proteus, cytofluorometric
determination of the nuclear DNA content also suggests
that part of the genome is replicated in interphase more
than once (Makhlin et al. 1979), and pulse-labelling of
this organism with [ 3 H]thymidine showed two waves of
nuclear DNA synthesis in different parts of the cell cycle
(Ord, 1968). However, it may be suggested that this
atypical mode of DNA replication reflects the perturbation of growth conditions, and may play a role in
adaptation to a changing environment, since in Acanthamoeba it was only found in cells cultivated in the
defined medium and in A. proteus, Goldstein & Prescott
(1967) found that all [3H]thymidine labelling took place
once within a cell cycle. In lower eukaryotic cells,
aberrant replication of DNA is induced when cell division is blocked and cell mass increases or, alternatively,
the gene concentration may become reduced when the
cell mass decreases (Berger, 1984). Thus, the abnormal
DNA replication in D cells may be related to the
imbalanced cell growth observed in these cultures. Estimates of cell cycle timing in Acanthamoeba differ considerably (Byers, 1979, 1986). These varying results may
be partly due to differences in the methodology; however, in the light of the present findings, they may also
result from different culture conditions.
The evidence found to date suggests that, in higher
eukaryotic cells, an important component in the alteration of cells that generate an overt cancer and/or in its
subsequent malignant progression is the relaxation or loss
of control of the number and timing of initiations of DNA
replication (Stark & Wahl, 1984; Stark, 1986; SchimkeeJ
al. 1986). It is interesting to note that in Acanthamoeba
an alteration in the timing of DNA replication is associated with an interference in normal developmental processes. ND cells that replicate only after the completion
of the cell cycle show a high encystation competence,
398
H. Jantzen et al.
whereas D cells that also replicate before mitosis are
constitutively committed to generation of trophozoites.
However, when D cells are cultivated in ND medium the
timing of DNA replication as well as the encystation
competence changes so that the degree of premitotic
DNA synthesis successively decreases and the percentage
of cells that become committed during the stationary
stage increases. This result indicates an incompatibility
between a cell cycle progression that includes premitotic
DNA replication and the developmental competence of
cells. This suggestion is consistent with the recent
finding that in Acanthamoeba, as in other unicellular
eukaryotic organisms (Weijer et al. 1984; Sharpe &
Watts, 1985; Nurse, 1985), the differentiation of cells is
related to a particular cell cycle phase. Synchronization
experiments revealed that this developmental decision
point is in late G2 phase of the Acanthamoeba cell cycle
(Stohr et al. 1987). Thus, these results indicate that,
owing to an improper timing of DNA synthesis, D cells
do not progress into late G2 phase of the cell cycle and
hence do not have the competence to become committed
to development.
The molecular mechanisms involved in developmental
commitment are not known in Acanthamoeba. We found
that those cells that express irregular DNA synthesis (D
cells or log stage ND cells in D medium) are in the noncommitted state, whereas premitotic DNA synthesis
cannot be induced in committed cells. This suggests that
the reduction in the capacity for irregular premitotic
DNA synthesis may be a prerequisite for development.
This work was supported by DFG grant II Bl-Ja 217/8-1 to
H.J.
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