Inhibition of DNA replication by fish oil-treated cytoplasm
is counteracted by fish oil-treated nuclear extract
Sybille
Rex1,2,
Maria A.
Kukuruzinska2, and
Nawfal W.
Istfan1
1 Department of Medicine, Section of Endocrinology,
Diabetes and Nutrition, Boston University School of Medicine, and
2 Department of Molecular and Cell Biology, Boston
University School of Dental Medicine, Boston, Massachusetts 02118
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ABSTRACT |
We have recently
noted that cells treated with fish oil and n-3-fatty acids show
slower DNA replication rates than cells treated with a control emulsion
or corn oil only. However, it is not clearly understood how
such an effect is induced. Fish oil and its metabolites are known to
have several modulating effects on signal transduction pathways.
Alternatively, they may influence DNA replication by interacting
directly with nuclear components. To investigate this problem in
greater detail, we have studied the kinetics of DNA synthesis in a
cell-free system derived from HeLa cells. Nuclei and cytosolic extract
were isolated from cells synchronized in early S phase after treatment
with control emulsion, corn oil, or fish oil, respectively. The nuclei
were reconstituted with cytosolic extract and a reaction mixture
containing bromodeoxyuridine (BrdU) triphosphate to label newly
synthesized DNA. The rate of DNA synthesis was measured by bivariate
DNA/BrdU analysis and flow cytometry. We show that fish oil-treated
cytosol inhibits the elongation of newly synthesized DNA by ~80% in
control nuclei. However, nuclei treated with fish oil escape this
inhibitory effect. We also show that addition of nuclear extract from
fish oil-treated cells reverses the inhibitory effect seen in the
reconstitution system of control nuclei and fish oil-treated cytosol.
These results indicate that polyunsaturated fatty acids can modulate
DNA synthesis through cytosolic as well as soluble nuclear factors.
n-3 fatty acids; cell cycle kinetics; flow cytometry; S phase; cell-free system
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INTRODUCTION |
ALTHOUGH THE
ASSOCIATION between dietary fat and cancer remains controversial,
several hypothetical mechanisms have been proposed to explain the
relationship between fat and cell proliferation (29, 32, 54,
69). Most of these mechanisms take into account the
participation of lipid molecules in signal transduction pathways (24, 26, 41, 46). Arachidonic acid (AA), an essential polyunsaturated fatty acid (PUFA) of the n-6 family, is a
component of cell membranes and a precursor of prostaglandin synthesis
in mammalian cells (5, 7, 23, 44, 52). Both AA itself and
one of its major metabolites, prostaglandin E2
(PGE2), play important roles in modulating the activity of
protein kinase C and the complex signaling pathway involving
mitogen-activated protein kinase (5, 7, 19-21, 23, 49,
52). Diets high in n-6 PUFAs have been generally associated
with increased cell proliferation, thus positively contributing to the
rate of cancer growth (50). On the other hand, diets rich
in n-3 fatty acids, such as fish oil, are often considered to be
inhibitory on cell proliferation due to reduction of AA in the plasma
membrane and the consequent decrease in PGE2. In fact,
several studies have associated fish oil with anti-cancer activity
(9, 15, 20, 21, 23, 28, 59).
Evidence that fish oil inhibits cell proliferation is derived
from experimental animal models as well as in vitro cell culture studies. Assuming that dietary fish oil reduces plasma membrane AA in
favor of n-3 PUFA, one expects a decrease in the transition of
proliferating G1 cells into S phase. This type of
regulation at the G1 restriction point stems from the
relationship among mitogenic signaling pathways, G1
cyclins, and the phosphorylation state of retinoblastoma protein and
characterizes the typical mechanism by which extracellular factors
affect cell proliferation (1, 2, 12, 33, 38, 48, 53,
56-58). However, studies in our laboratory in fish
oil-treated cultured cells in conjunction with 5-bromo-2'-deoxyuridine
(BrdU) pulse labeling and bivariate BrdU/DNA flow cytometry suggested
that the G1 to S phase transition was not affected (present
study and Ref. 35). Instead, fish oil-treated cells were
more typically characterized by a longer S phase duration despite a
normal G1 to S phase transition. Because DNA replication in
eukaryotic cells starts at multiple sites called replication origins
(22, 60), we proposed that fish oil treatment interfered
with the spatial and/or temporal organization of these replication
origins in a manner causing S phase lengthening. In support of this
hypothesis, we were able to document a spatial change in the location
of the well-characterized replication origin, ori-
, in the
dihydrofolate reductase locus in exponentially growing, fish
oil-treated Chinese hamster ovary (CHO) cells (35).
To determine whether fish oil exerted its effect directly on the
replicating nucleus or through signaling pathways mediated by the
cytoplasm, we used a replicating cell-free system derived from HeLa
cells (40). In this system, nuclei separated from synchronized HeLa cells and reconstituted with a cytosolic extract can
be induced to start DNA replication (40). Therefore, if fish oil acted directly on the nucleus, we expected to observe a
reduced rate of DNA replication in the nuclei derived from fish oil-treated cells, regardless of the source of the cytosolic extract. In the present study, we measured DNA replication by using BrdU pulse
labeling and bivariate DNA/BrdU flow cytometry in reconstituted HeLa
systems where the individual components were derived from either
oil-treated or control cells. For comparison, we also analyzed cell
proliferation kinetics in fish oil-treated HeLa cells by using a
similar flow cytometric method. Separate controls based on growth media
supplemented with either egg phosphatidylcholine or corn oil were
included for both the whole cell and the reconstituted system. Our
results clearly indicate that fish oil exhibited a strong inhibitory
effect that was mediated through the cytosolic extract. Interestingly,
fish oil-treated nuclei, but not control nuclei, were able to escape
this inhibitory effect on DNA replication, suggesting that fish
oil-treated nuclei can counterbalance the inhibitory effect of fish
oil-treated cytosol.
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MATERIALS AND METHODS |
Preparation of Oil Emulsions
The preparation of oil emulsions followed mainly a procedure
described by Fox and DiCorleto (27). Briefly, fish oil or
corn oil (General Nutrition), respectively, was dissolved in chloroform to which 5% (wt/wt) L-
-phosphatidylcholine from egg
yolk (eggPC; Sigma) and 0.03% (wt/wt) butylated hydroxytoluene (as
antioxidant) were added. The solutions were dried under a nitrogen
stream, placed under vacuum overnight to remove residual solvents, and resuspended in phosphate-buffered saline (PBS) at an oil concentration of 15 mg/ml. To prepare a control solution, only eggPC and butylated hydroxytoluene were mixed and dried; PBS was added at the same ratio as
for the oil emulsions.
Cell Culture and Oil Treatment
HeLaS3 cells (American Type Culture Collection) were cultured as
exponentially growing monolayers on 100-mm cell culture plates in F-12K
medium supplemented with 10% fetal calf serum, 108 U/ml penicillin,
and 0.11 mg/ml streptomycin (all GIBCO-BRL).
Oil emulsion or a corresponding volume of control solution was added to
exponentially growing cells at a final oil concentration of 150 µg/ml. Medium and emulsion were replaced daily, and the treatment was
continued until the experiment was performed.
Experimental Part I: Cell Cycle Kinetics
Growth characteristics.
HeLa cells were plated on day 0 and then treated for 5 consecutive days with control, corn oil, or fish oil emulsion. On
day 5 of the treatment, the cells were harvested and then
reseeded at 1-2 × 106 cells per plate. For the
following 4 days, the treated cells were counted every 20-28 h by
use of a hemacytometer and trypan blue (GIBCO-BRL). At each time point,
cells were aliquoted in triplicates and each triplicate sample was
counted two to three times. The growth curve for each treatment group
was determined from three separate experiments. The rate of exponential
cell growth (first-order rate constant Kg) was
derived from semilogarithmic growth curves by regression analysis, and
the actual doubling time (Td) was calculated
from the following equation
|
(1)
|
BrdU pulse-chase labeling procedure.
For kinetic analysis of the cell cycle, HeLa cells were grown at 37°C
in the same manner as described for the growth curves. On day
5, cells were reseeded, and treatment was continued for one more
day to reestablish exponential growth. Cells were then pulse-labeled
for 1 h with BrdU (Sigma) at a final concentration of 10 µM.
Subsequently, cells were either harvested immediately (0 h time point)
or chased for 4 h with fresh BrdU-free medium containing the
corresponding concentration of control or oil emulsion. After
harvesting, cells were fixed in 70% cold ethanol and stored at
20°C until further processing for flow cytometric analysis as
described below.
Double-staining procedure and flow cytometry of labeled cells.
Fixed cells were double-stained for DNA and BrdU content by a standard
protocol as previously described (35). Briefly, fixed cells were washed with PBS and treated with 2 N HCl containing 0.5%
(vol/vol) Triton X-100 (Sigma) for 1 h at room temperature to
denature the DNA and improve antibody binding. Subsequently, cells were
neutralized with 0.1 M sodium tetraborate (pH 8.5) and washed twice
with PBS containing 0.5% (vol/vol) Tween 20 and 1% (wt/vol) bovine
serum albumin (wash solution). After pelleting, cells were resuspended
in 50 µl of wash solution, and then 15 µl of FITC-conjugated
monoclonal anti-BrdU antibodies (PharMingen) were added and cells
were incubated for 1 h in the dark. To stain for DNA content,
cells were then washed with PBS and incubated in the dark for 1 h
with 20 µg/ml propidium iodide (PI; Sigma) in PBS.
Afterwards, the double-stained cells were analyzed with a FACScan flow
cytometer (Becton Dickinson) at an excitation wavelength of 488 nm and
a laser power of 15 mW. The red fluorescence from PI was collected
through a 585-nm band-pass filter, and the green fluorescence from
FITC-labeled anti-BrdU antibodies was collected through a 530-nm
band-pass filter. The red fluorescence was calibrated by adjustment of
the G0/G1 peak to a fixed channel
number. Data from 105 cells were recorded in a 1,024 × 1,024-channel distribution showing the linear amount of total DNA
(red) and the logarithmic amount of BrdU (green) by using the software
CellQuest (Becton Dickinson).
Kinetic analysis of flow cytometric data.
To characterize the cell cycle kinetics, the recorded flow cytometric
data were analyzed by using two different methods. First, we determined
the distribution of cells in each phase of the cell cycle by
mathematical analysis of the DNA histograms (red fluorescence only)
using the computer program ModFit (Verity Software House, Topsham, ME).
Second, we determined the progression through the cell cycle by
analysis of the bivariate contour plots derived from the double-stained
cells (red and green fluorescence; see e.g., Fig. 2) using the software
program IsoContour (Verity Software House) as described below.
In DNA/BrdU contour plots (see e.g., Fig. 2), the populations of cells
in G0/G1 and G2/M
phase as well as the population of BrdU-labeled cells were gated and
the mean red fluorescence of each population (FG1,
FG2, and FL, respectively) was determined. The
position of BrdU-labeled cells relative to cells in G1 and G2 phase, formally defined as relative movement (RM), was
calculated according to the following equation (6)
|
(2)
|
where RM(t) is the relative movement of BrdU-labeled
cells measured at either 1 or 5 h following BrdU pulsing.
After a 4-h chase with BrdU-free medium, the BrdU-labeled cells could
be separated into labeled divided (fld) and labeled undivided (flu) subgroups, respectively (see Fig. 2) and
were quantified by gated analysis. On the basis of these fractions, a
cell cycle parameter
was calculated according to the definition of
White et al. (68) by using the following equation
|
(3)
|
This parameter (
) is related to the potential doubling time
(Tpot) and the DNA synthesis time
(TS) by the following relationship
|
(4)
|
The parameter was subsequently used to calculate the cell
production rate (c), according to the following equations,
as previously described by White et al. (67)
|
(5)
|
where
|
(6)
|
and t is the time interval between BrdU labeling and
flow cytometric analysis. After c was estimated,
TS, Tpot, and the time spent in G2/M phase (TG2+M)
were derived from the following relationships (67)
|
(7)
|
|
(8)
|
|
(9)
|
Finally, an estimate of cell loss was derived from comparison of
Tpot and Td according to
the following equation for the cell loss factor (
), expressed as a
fraction (62)
|
(10)
|
Experimental Part II: Cell-Free Reconstitution Experiments
Synchronization in S phase.
In preparation of a reconstitution experiment, culture media were
supplemented with an oil or control emulsion for 4 days. Cells were
reseeded on day 5, and the treatment with either emulsion was continued. On day 6, exponentially growing cells were
synchronized in S phase by a single thymidine block for 24 h. A
final concentration of 2.5 mM thymidine (Sigma) was found to be
sufficient for synchronization of HeLa cells in early S phase.
Subsequently, cells were harvested and processed as described below.
The adequacy of the synchronization procedure was verified by flow
cytometry. Briefly, fixed synchronized cells were washed with PBS,
incubated with 0.5 mg/ml RNase A in PBS for 2 h at room temperature, washed with PBS, and subsequently incubated with 20 µg/ml PI in PBS for several hours or overnight. Flow cytometric analysis of DNA histograms was performed as explained for
Experimental Part I.
Preparation of nuclei and cytosolic extracts.
A procedure described by Krude and colleagues (39, 40) was
followed with minor modifications. Briefly, cell monolayers were washed
once with ice-cold Hanks' balanced salt solution (HBSS). After further
addition of 2 ml of ice-cold HBSS, cells were scraped off the
substratum at 4°C and centrifuged at 1,000 g for 10 min in
an MR18.22 centrifuge (Jouan). The pellet was resuspended and swollen
in hypotonic buffer (10 mM HEPES, pH 7.8, 10 mM KCl, 1.5 mM
MgCl2, and 2 mM DTT) for 15 min on ice. Cells were
disrupted with 50-100 strokes in a Dounce homogenizer (pestle B).
Nuclei were checked for the presence of undisrupted cells by using
trypan blue. Accordingly, the homogenization was either continued or terminated. After centrifugation for 5 min at 1,800 g, the
pellet (nuclei) was separated from the supernatant (cytosolic
fraction), washed with PBS, and counted with a hemacytometer. About
1.5 × 106 nuclei per Eppendorf tube were spun down
and resuspended in 50-100 µl of storage solution [250 mM
sucrose, 75 mM NaCl, 0.5 mM spermine tetrachloride, 0.15 mM spermidine
trichloride (both Sigma), and 3% (wt/vol) bovine serum albumin] and
stored at
80°C. The cytosolic fraction was kept on ice until
further centrifugation at 20,000 g for 30 min. The protein
concentration ranged between 3 and 12 mg/ml. Subsequently,
the supernatant was stored at
80°C.
Preparation of nuclear extracts.
Nuclear extracts were prepared freshly before use from isolated nuclei
as reported by Krude et al. (40). Pelleted nuclei were
resuspended at a concentration of 150 × 106 nuclei/ml
in hypotonic buffer containing a final concentration of 0.4 M NaCl.
After extraction for about 2 h on ice and vortexing every 30 min,
the suspension was centrifuged for 30 min at 16,000 g at
4°C. The supernatant (nuclear protein concentration of 7-11 mg/ml) was immediately separated from the pellet and kept on ice.
Reconstitution experiments.
About 1.5 × 106 nuclei synchronized in early S phase
were used per reconstitution experiment. Nuclei in storage solution
were pelleted and resuspended on ice in a reaction mixture composed of
5× reaction buffer and S phase cytosolic extract (containing 200-300 µg of protein per 1-1.5 × 106
nuclei). The amount of reaction buffer added was adjusted so that the
total volume of the reaction solution yielded a final concentration of
40 mM HEPES, pH 7.8, 7 mM MgCl2, 3 mM ATP, 0.1 mM each of
GTP, UTP, and CTP, 0.1 mM each of dATP, dGTP, and dCTP, 0.5 mM DTT, 40 mM creatine phosphate, 5 µg of phosphocreatine kinase (both
Boehringer-Mannheim), and either 0.1 mM BrdU triphosphate (BrdUTP;
Sigma) or dTTP. The reconstitution reaction was started by transferring
the samples from ice to 37°C. The nuclei were pulse-labeled for 30 min with reaction buffer containing BrdUTP. Subsequently, nuclei were
either fixed immediately (0 h time point) or spun down, resuspended in
fresh reaction buffer containing dTTP, chased for 2 h at 37°C,
and then fixed (2 h time point). In all reconstitution experiments, the
chase time was limited to 2 h because nuclear integrity became
visibly compromised afterwards.
In reconstitution experiments with nuclear extracts, 100-130 µg
of nuclear protein extract, 180-250 µg of cytosolic extract (both from cells treated with fish oil), and reaction buffer were added
to 1.5 × 106 nuclei from control cells, and nuclei
were then BrdUTP-labeled and chased as explained above. Nuclei were
stained with PI and FITC-conjugated anti-BrdU antibodies and analyzed
by flow cytometry in the same manner as the cells.
Statistical Analysis
Statistical comparison of multiple groups was evaluated by
one-way analysis of variance (ANOVA). Subsequently, specific
comparisons between two means were made by the Student's
t-test only when ANOVA showed significant overall difference
(P < 0.05). Comparison of data derived from two
separate means, such as
RM, was made by a modified Student's
t-test, known as "Welch's approximate t" as
described by Zar (70). All statistical analyses were
performed by using the software programs Excel and SigmaPlot.
 |
RESULTS |
Fish Oil Treatment Decreases the Proliferation Rate of HeLa Cells
To ensure exponential growth conditions, we determined the growth
curves for all three treatment groups. After 5-day treatments with
eggPC, corn oil, and fish oil, cells were reseeded and grown for four
consecutive days in control or oil-containing medium. Daily aliquots
were used to determine mean cell numbers, as summarized in a
semilogarithmic plot in Fig.1. Whereas
control and corn oil-treated cells showed very similar growth rate
constants (Kg, slope), fish oil-treated cells
exhibited a smaller value of KG. The actual doubling times (Td) were determined from the
slope of each straight line according to Eq. 1, and the
values are given in Table 1. Fish
oil-treated cells exhibited a significant increase in
Td (29.3 ± 1.5 h) by ~30% compared
with control (22.1 ± 0.3 h) or corn oil-treated cells
(22.8 ± 1.8 h), respectively (P
0.05 for fish
oil vs. control and corn oil).

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Fig. 1.
Logarithmic plot of growth curves showing the number of
cells as a function of time for cells treated with
L- -phosphatidylcholine from egg yolk (eggPC; control),
corn oil, or fish oil. Each data point is an average of 3 individual
samples, whereas each sample was counted 2-3 times. Values
represent means ± SE. Continuous lines show the best linear fit
from which first-order growth rate constants KG
were derived (0.031/h for eggPC and corn oil, 0.024/h for fish oil).
Growth curves were measured 3 times in total, whereas 1 representative
set is shown here.
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Fish Oil Treatment of HeLa Cells Increases the S Phase Duration
Time Without Affecting G1/S Phase Transition
First, DNA histograms were analyzed to determine the effect of oil
treatment on the distribution of cells within the cell cycle. Results
of the percentages of cells in each cell cycle phase are summarized in
Table 2, showing no significant effects for either oil treatment on cell cycle distribution. Table 2 also shows
that the labeling index, expressed as the percentage of BrdU uptake in
cells after 1 h of labeling time, was also similar among the three
treatment groups. However, significant differences were noted in cell
cycle kinetic parameters derived from BrdU labeling and bivariate flow
cytometry, as explained in MATERIALS AND METHODS. Cell
cycle data were obtained by analyzing contour plots
(Fig.2) from fish oil- and corn
oil-treated cells recorded at 1 h (A and C)
and 4 h (B and D), respectively, after BrdU
treatment. As noted in Fig. 2, the population of BrdU-labeled cells can
be divided into labeled undivided (flu) and labeled divided
fractions (fld). These results are summarized in Table
3. Whereas control and corn oil-treated
cells show 2.8 ± 0.2 and 3.1 ± 0.3% of cells, respectively, in the fld fraction, fish oil-treated cells
have a significantly lower percentage of cells in this fraction
(1.1 ± 0.2%) (P < 0.0005 for fish oil vs.
control and corn oil). However, after 4 h of chase time, the
number of cells in the flu fraction had not changed
significantly for all three treatment groups. On the basis of these
results, the parameter
(Eq. 3), which represents the
fraction of cells traversing the S phase during one cell cycle, did not
vary significantly between the corn oil and fish oil treatment groups.
These findings imply equal rates of G1 to S phase
transition in HeLa cells treated with fish oil, corn oil, or eggPC.

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Fig. 2.
Contour plots of bivariate BrdU/DNA distribution obtained by flow
cytometric analysis of HeLa cells treated with fish oil (A
and B) or corn oil (C and D), respectively.
Cells were pulse-labeled for 1 h with 10 µM BrdU and immediately
fixed (0 h chase, A and C) or chased for 4 h
with BrdU-free medium (B and D). The
x-axis represents the red fluorescence channels (linear
scale), which correspond to the total DNA content [stained with
propidium iodide (PI)]. The y-axis represents the green
fluorescence channels (logarithmic scale), which correspond to the
extent of incorporated BrdU (stained with FITC-conjugated BrdU
antibodies). The positions of cells in
G0/G1, S, and G2/M
phases are indicated. After a chase of 4 h, populations of labeled
divided and labeled undivided cells can be distinguished.
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Estimates of the DNA synthesis time (TS) were
derived from the relative movement (RM) of BrdU-labeled cells and the
parameter
according to the method of White et al.
(67). As noted in Table 3 and Fig.
3, RM was similar in the treatment groups
1 h after BrdU pulse labeling. However, cells analyzed 4 h
after BrdU labeling showed significantly lower RM values in the fish oil-treated group compared with the other two groups (P < 0.00005 for fish oil vs. control and corn oil; Table 3 and Fig. 3).
Based on these values and Eqs. 5-7, the mean DNA
synthesis time was determined as 9.88 ± 0.25 h in fish
oil-treated cells, 7.29 ± 0.32 h in control cells, and
6.90 ± 0.39 h in corn oil-treated cells, respectively (P < 10
5 by ANOVA). Similarly, estimates
of the potential doubling times (Tpot) were
significantly longer in the fish oil-treated cells, whereas estimates
of the time spent in G2/M phase were remarkably similar in all treatment groups (Table 1). Finally, estimates of cell
loss, also summarized in Table 1, were not significantly different
among the three treatment groups. These findings implicate a slower S
phase progression as the main cause of decreased cell proliferation
rate in fish oil-treated HeLa cells.

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Fig. 3.
The relative movement of HeLa cells treated with eggPC
(control), corn oil, or fish oil was calculated according to Eq. 2 from contour plots as shown in Fig. 2. Each data point is
averaged over 4-5 experiments. Values represent means ± SE.
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Cytoplasm From Fish Oil-Treated Cells Inhibits DNA Synthesis in
Nuclei From Control Cells But Not in Nuclei From Fish Oil-Treated Cells
To differentiate between cytosolic and/or nuclear sites of action
for fish oil on DNA replication, we performed a set of reconstitution experiments using a cell-free system prepared from control, corn oil-treated, and fish oil-treated cells. In this system, S
phase-synchronized HeLa cells (see e.g., Fig.
4) were used to prepare nuclei and cytosolic extracts as described in MATERIALS AND METHODS.
In all, seven cell-free reconstitution systems were included in this
study, as summarized in Tables 4 and
5. As expected, ~90% of the nuclei were in S phase, with a small percentage in
G0/G1 and G2/M phase as depicted in Fig. 5. Thus it was
possible to determine the RM of BrdU-labeled nuclei in the
reconstituted system. Typical bivariate plots of reconstituted
double-stained nuclei are shown in Fig. 5, A (0 h) and
B (2 h). Because of the density of nuclei in the early part
of S phase, it was not possible to discern movement of nuclei along the
DNA axis after 2 h by simple visual inspection. However,
actual movement is more readily seen in the relative distribution of
nuclei along the DNA axis, as demonstrated in Fig. 5C, which
is shown for illustrative purposes. In Fig. 5, A and
B, the DNA axes were divided into six discrete S phase
compartments in units of RM (<0.35, 0.35-0.50, 0.50-0.65,
0.65-0.80, 0.80-0.95, 0.95-1.0), and the percentage of
nuclei in each compartment was determined. As expected, the majority of
nuclei synchronized in S phase were found in the compartment with
RM < 0.35 at 0 h. Two hours after reconstitution, the
relative number of nuclei with RM < 0.35 diminished from
64.5 ± 1.6% to 56.6 ± 1.0% (P < 0.001), whereas the percentage of nuclei in the compartment of 0.35 < RM < 0.50 significantly increased from 26.1 ± 1.7% to
34.0 ± 1.9% (P < 0.005). Similarly, there were
significant increases in the relative number of cells in higher RM
compartments after 2 h of reconstitution (Fig. 5C).
These results, showing a redistribution of BrdU-labeled nuclei from
lower to higher RM compartments, indicate a significant and measurable
increase in DNA content in the reconstituted nuclei.

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Fig. 4.
DNA histograms presenting the counts per channel vs.
channel number for untreated asynchronous HeLa cells (A) and
for cells synchronized in early S phase by a 24-h thymidine block
(B).
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Fig. 5.
Contour plots of bivariate BrdU/DNA distribution were obtained from
flow cytometric analysis of nuclei isolated from control cells
synchronized in early S phase and reconstituted with cytosol from
control cells (group 1) (see Table 4 and Fig. 6). The nuclei
were pulse-labeled for 30 min with 100 µM BrdU triphosphate (BrdUTP)
and either immediately fixed (A) or chased for 2 h with
dTTP (B). The notation of both axes is the same as in Fig.
2. The positions of nuclei in G0/G1, S,
and G2/M phase, respectively, are indicated.
C: for illustrative purposes and to give evidence of
relative movement (RM) among the S phase nuclei, the RM values were
grouped into 6 compartments (<0.35, 0.35-0.5, 0.5-0.65,
0.65-0.8, 0.8-0.95, 0.95-1.0). For each compartment the
percentage of BrdU-labeled nuclei was determined at 0 and 2 h.
*Statistical significant difference between both time points.
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The relative rate of DNA synthesis in a reconstituted system can be
assessed from the change of RM over the 2-h time period. Values of RM
for the total S phase population at each reconstitution time point are
summarized in Tables 4 (fish oil) and 5 (corn oil) for all treatment
groups. In Table 4, RM at 0 h was not significantly different
among the four treatment groups (by ANOVA). However, significant
differences were noted in RM measured at 2 h
(P < 10
5; by ANOVA) with the
reconstitution group of fish oil-treated cytosol/control nuclei
(group 3) having the smallest value (P < 0.01 for group 3 vs. groups 1, 2, or
4; by t-test). A summary of the relative rates of
DNA synthesis in each of the treatment groups, expressed by the 2-h
increment in RM (
RM), is presented graphically in Fig.
6. As noted in Fig. 6, fish oil-treated
cytosol appears to reduce the relative rate of DNA synthesis by ~80%
in control (untreated) nuclei (P < 0.01 for
group 3 vs. group 1). In contrast, when fish
oil-treated nuclei were reconstituted with fish oil-treated cytosol
(group 2), no such inhibition in DNA synthesis was noted.
Similarly, there was no inhibition of DNA synthesis in the fish
oil-treated nuclei reconstituted with control (untreated) cytosol.

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Fig. 6.
The change in relative movement ( RM) is displayed on a
bar graph for all reconstitution systems investigated. RM was
determined after a BrdUTP pulse and a subsequent dTTP chase for 2 h. Values represent means ± SE. A: reconstitution of
fish oil-treated and/or control nuclei and cytosol. A detailed
description of these treatment groups is given in Table 4. Results are
averaged over 6-12 experiments per group. *Group 3 is
statistically significant compared with the other groups in A
(P < 0.01). B: reconstitution of corn
oil-treated and/or control nuclei and cytosol. A detailed description
of these treatment groups is given in Table 5. Results are averaged
over 6-9 experiments per group. C: reconstitution
including nuclear extracts from fish oil-treated cells. A detailed
description of these treatment groups is given in Table 6. Results are
averaged over 5-6 experiments per group. *Group 8 is
statistically significant compared with other groups in C.
Refer to text for details.
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In the corn oil-treated groups (Table 5), differences were noted in RM
at 0 h (P < 0.0001; by ANOVA) as well as at
2 h (P < 0.005; by ANOVA). However, comparison of
RM among the reconstitution systems of the corn oil treatment group
(Fig. 6B) showed no significant differences except for
group 7 (
RM = 0.087 ± 0.008) compared with
group 1 (
RM = 0.054 ± 0.007)
(P < 0.01). Thus nuclei from corn oil-treated cells
appear to progress faster through S phase than nuclei from control
cells when both are incubated with control cytosol.
Nuclear Extract From Fish Oil-Treated Cells Reverses the Inhibitory
Effect of Fish Oil-Treated Cytosol
To further establish the enhancing effect of nuclear components on
the DNA synthesis in the fish oil reconstitution system, we prepared
nuclear extract from fish oil-treated cells and incubated the nuclei
derived from control cells with fish oil-treated cytosol and fish
oil-treated nuclear extract (group 8). Results of RM at 0 and 2 h as well as
RM are presented in Table
6 and Fig. 6C, with pertinent
comparison groups. The addition of nuclear extract reversed the
inhibitory effect observed with fish oil-treated cytosol and control
nuclei (group 3), leading to an increase in
RM from
0.013 ± 0.004 for group 3 to 0.099 ± 0.023 for
group 8 (P < 0.001). We also note that
RM for group 8 was larger than that for group
2, where fish oil-treated nuclei were incubated with fish
oil-treated cytosol.
 |
DISCUSSION |
This study was conducted to further clarify the mechanism(s)
by which fish oil alters the proliferation of mammalian cells. The
relevance of this inhibitory effect of fish oil for the relationship between diet and cancer has been addressed in the literature. In fact,
both fish oil supplementation and pharmacological treatment aiming at
the inhibition of cyclooxygenase activity to alter the prostaglandin
composition of cells have been proposed in cancer prevention (13,
17, 44, 51, 54). For the most part, fish oils are thought to
induce changes in cell function (3, 16, 23, 27, 28, 36, 45, 47,
55, 61, 65) by replacing membrane n-6 fatty acids, such as
AA, with PUFAs of the n-3 family. Consequently, multiple changes
in plasma membrane function, such as transmission of receptor-induced
extracellular signals to the interior of the cell, have been
hypothesized (4, 14, 19, 23, 31, 42, 63-65).
We have shown previously that addition of fish oil to the diet of
tumor-bearing rats reduces the growth rate of tumors and causes a
prolongation of the time required for DNA replication without affecting
the transition of proliferating cells from G1 to S phase
(36). These findings suggested that fish oil might interact with the DNA replication machinery at the level of the replicating nucleus separately from its putative downregulatory effect
on classic mitogen-induced signaling pathways, as had been proposed
(10, 33, 53). In support of this hypothesis, we recently
demonstrated a change in the spatial localization of the
well-characterized ori-
replication origin in CHO cells
(35).
Our main objective in the present study was to determine whether fish
oil alters DNA replication by a direct effect on the nucleus and
whether cytoplasmic signaling is also involved in this inhibitory
effect. For this purpose we used a cell-free DNA replicating system
derived from HeLa cells that was first described by Krude et al.
(40). The system allows reconstitution of control or fish
oil-treated cytoplasm with either control or fish oil-treated nuclei in
a crossover design. As shown by Krude et al. (40), synchronized G1 nuclei separated from HeLa cells can be
induced to assemble the DNA replication machinery if incubated in the presence of a cytosolic extract, a nuclear extract from S phase cells,
and an energy-generating system. These reconstituted cell components
engage in DNA synthesis for several hours. In the present study, we
used S phase nuclei that had already assembled the DNA replication
machinery, thus obviating the need for separate nuclear extracts in the
reconstitution system. Before cell fractionation, these nuclei were
exposed to fish oil in the growth medium for 5 days, a period
sufficient to induce changes in S phase progression, as noted in
MATERIALS AND METHODS. Therefore, if fish oil affected the
assembly of replication origins by a direct effect on nuclear organization, we expected to detect differences in S phase progression in the reconstituted system composed of fish oil-treated nuclei and
cytosolic extract from control cells. Similarly, the experimental design allowed testing of the possibility that fish oil mediates its
effects through the cytoplasm if DNA replication is affected in the
system reconstituted from fish oil-treated cytosol and control nuclei.
In the absence of prior information about the effect of fish oil on
proliferation kinetics of HeLa cells, fish oil treatment of whole HeLa
cells was necessary in the present study. This set of experiments was
performed under conditions of exponential growth, in line with our
previous characterization of the effect of fish oil in other cell lines
(35). As reported here, initial studies utilizing whole
HeLa cells depict a similar response to fish oil supplementation as
previously noted in CHO cells (35). After 5 days of
treatment with fish oil-containing medium, HeLa cells grow at a
significantly slower exponential rate. This difference in actual cell
proliferation rate is not explained by an increase in cell death,
because flow cytometric data did not reveal evidence of
apoptosis or cell fragmentation as in our previous studies with
individual n-3 fatty acids (11). It also is not
explained by a reduction in the entry of proliferating cells into S
phase, as depicted by the kinetic parameter
. Only the estimate of
DNA replication time, derived from the rate of progression of
BrdU-labeled cells relative to the position of cells in
G0/G1 and G2/M phase, was significantly increased in fish oil-treated cells (Table 1). As
noted in Table 1, the increase in Tpot is
predominantly explained by a longer S phase duration in fish
oil-treated cells.
In the reconstituted system, DNA synthesis takes place within 2 h,
as noted in the increase in relative movement and in redistribution of
BrdU-labeled nuclei toward higher DNA content (Fig. 5). However, the
stability of this system becomes compromised after 2 h, thus making it impossible to determine the kinetics of DNA synthesis in the
later stages of S phase. For this reason, it was not possible to obtain
estimates of TS, Td, or
other cell cycle kinetic parameters from the reconstituted system.
Despite this limitation in the cell-free HeLa system used in the
present study, our results clearly demonstrate an inhibitory effect on
DNA replication mediated by the fish oil-treated cytoplasm. Our
findings also suggest that this cytosolic inhibitory signal on DNA
replication caused by fish oil treatment is counterregulated at the
level of the nucleus. Interestingly, nuclear extracts prepared from
fish oil-treated HeLa cells can completely restore DNA synthesis in the
inhibited reconstitution system. Therefore, escape from DNA synthesis
inhibition and completion of the S phase in the 5-day fish oil-treated
HeLa nuclei is not achieved by an alteration in nuclear structure or chromatin organization but, rather, by a soluble nuclear factor.
Although the effects of fish oil on cell proliferation are likely to be
related to its content of n-3 fatty acids, this conclusion cannot
be ascertained from the current study. We have included an experimental
group treated with corn oil to evaluate for the effect of n-6
PUFA. Assuming that n-3 fatty acids ultimately account for the
inhibitory effect, one may expect an opposite stimulatory effect if
n-6 fatty acids are introduced into the system. The results of the
current study are consistent with this possibility, because corn
oil-treated nuclei appear to synthesize DNA at a faster rate than
control nuclei. However, further studies to determine the significance
of potential changes in cytoplasmic signaling and nuclear response, in
relationship to n-3 and n-6 fatty acid cellular composition,
are needed.
The mechanisms of DNA replication inhibition and those of the apparent
recovery of the fish oil-treated nucleus in this study are only
speculative at this time. DNA replication initiates at specific
chromosomal sites in a complex process that remains incompletely understood in eukaryotic cells (18, 22, 25, 34, 66). The
fact that the nuclei used in this study were already in the early S
phase at the time of reconstitution implies that the inhibitory effect
of fish oil occurred during DNA elongation. Individual steps that could
potentially contribute to the total effect of fish oil include strand
separation by helicase and priming, as well as actual polymerase
activity. It is also possible that the assembly of replication
initiation complexes at late replication origins, which are activated
in later stages of the DNA replication process, does occur in early S
phase and that this may be specifically prevented by the fish
oil-treated cytosolic extract. Alternatively, slowing of DNA
replication in fish oil-treated cells may result from activation of the
regulatory intra-S phase checkpoint system (8, 30, 37,
43). However, it is important to note that this system normally
responds to DNA damage, for which we have no evidence in the current
study. Furthermore, this mechanism does not explain the apparently
normal DNA synthesis response in the fish oil-treated nuclei
reconstituted with fish oil-treated cytosol. Obviously, further
research is needed to clarify these numerous possibilities.
In conclusion, we present here evidence that fish oil treatment of
proliferating mammalian cells triggers an inhibitory signal for DNA
synthesis in the cytoplasm. It also appears that counterregulatory mechanisms can occur within the fish oil-treated nucleus to overcome this inhibition in the whole cell. Further investigations of the components of this system will be helpful in understanding the interaction among fatty acids, fats, and cell proliferation.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Cancer Institute Grant CA-45768
(to N. W. Istfan).
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
N. W. Istfan, Boston Univ. School of Medicine, Section of
Endocrinology, Diabetes and Nutrition, 88 East Newton St., Evans 201, Boston, MA 02118 (E-mail:
nsteph3{at}cs.com).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 17, 2002;10.1152/ajpcell.00121.2002
Received 14 March 2002; accepted in final form 11 June 2002.
 |
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