[CANCER RESEARCH 37, 482-489, February 1977]
The Relationship between Polyamine Accumulation and DNA
Replication in Synchronized Chinese Hamster Ovary Cells
after Heat Shock
Eugene W. Gerner and Diane Haddock Russell2
University of Arizona College of Medicine, Departments of Pharmacology and Radiology (D. H. R.J/Division of Radiation Oncology fE. W. G.J,
Tucson, Arizona 85724
SUMMARY
INTRODUCTION
The relationship of pobyamine accumulation and semi
conservative DNA replication was studied in synchmanaus
Chinese hamster ovary cultures, progressing through the
cell cycle either normally at 37°amafter hyperthermic expo
sure (43°for1 hr) during G1amS phase. In control cultures,
intracellular pobyamine levels decreased as cells divided
and then meaccumulated as cells exited G1 and proceeded
through the S and G2 phases. Immediately after cultures
were exposed to 43°heat for 1 hr in G1 phase, intracellular
levels of spermidine and spemminewere reduced compared
to controls. Coordinate with the depletion of the intraceblu
banlevels of these polyamines following exposure at 43°,
extracellulan bevels of spermidine and spermine were in
creased. The ratio of intracellubamto extracellulam amounts
of bath these polyamines changed from 1 to 1.5 to approxi
mateby 0.2 to 0.3 after hyperthermic exposure. These cub
tunes exposed to 43°heat during G1 initially showed de
pressed bevels of replicated DNA, but near-control matesof
DNA replication were attained in a temporally related man
ner with the meaccumulation of intracellular spermidine and
spermine levels. When cultures were exposed to 43°heat in
S phase, intracellular amounts of spermidine and spermine
were again reduced , and increased extraceblular levels of
these polyamines were observed . In these S-phase-treated
cultures, cells were able to continue replicating their DNA
but at a much reduced matecompared to controls. These
results and others show that: (a) exposure of cells at 43°
causes a depletion of intracellular levels of spermidine and
spenmine, suggesting that an immediate aspect of thermal
damage is a membrane defect that markedly affects the
transport of these molecules across cell membranes; (b)
exposure of either G1- or S-phase cultures to 43°heat
caused a depression of bulk DNA-synthetic matesresulting
in a prolongation of S phase@and (c) the intracellular reac
cumulation of spemmidine and spemminefollowing exposure
of G1 cells to a 43°heat shock is temporally related to the
recovery of near-normal DNA synthetic rates in these cells.
The potential clinical importance of hyperthenmia as an
anticancer agent, used either alone on as an adjuvant to
radiation on chemotherapeutic drugs, derives from an ex
tensive number of reports in both the scientific and medical
literature dating back to the last century (see Refs. 7, 17,
and 27 for current reviews). It is now well documented that
exposure of cells in culture to temperatures above 41°me
suits in a lass of proliferative capacity (6, 10, 30). An in
creasing number of reports suggest that cancer cells may
be more sensitive to thermal killing than normal cells (2, 13).
However, the biochemical nature of hyperthermia-in
duced lass of cellular proliferative capacity is still poorly
understood. We have chosen to study polyamine biosyn
thesis after heat shock because of previous work which has
presented strong evidence demonstrating a close commela
tion between intracellular polyamine levels and cell growth
in vivo and in vitro (3, 11, 20, 22, 23) and because the
specific steps in polyamine biosynthesis are fairly well Un
derstood. Intracellular spemmidine levels have been shown
to correlate well with RNA content (3, 4, 19), while spermine
seems to parallel DNA amounts (24). Thus, the observation
that hyperthermic exposure inhibits RNA (26) and DNA syn
thesis (16, 18) lends further rationale to the study of palya
mines in order to understand the mechanisms of heat
shock-induced loss of cellular proliferative potential.
In this study, the effects of hyperthenmia an polyamine
biosynthesis and accumulation are compared with parallel
studies measuring matesof semiconservative DNA replica
tian, which we have used as a measure of cell cycle pro
gression. Our results suggest that the major immediate
effects of hyperthemmia on cell progression may derive from
a cell membrane defect resulting in the lass of molecules,
including the polyamines, which are necessary fan normal
cell growth rates.
1 This
Society
work
was
supported
by Grant
and by USPHS Grants
IN-lb
CA-18273,
from
cA-14783,
the
American
CA-17343,
Cancer
and CA
17094.
2 Recipient
of
Research
Career
Development
Award
CA-00072
National Cancer Institute.
Received December 29, 1975; accepted November 5, 1976.
482
from
MATERIALS AND METHODS
Cell Culture Procedures. CHO3 cells were grown as man
olayen cultures in McCoy's 5A Medium supplemented with
20% fetal calf serum (both from Grand Island Biological Co.,
Grand Island, N. Y.). The growth medium also contained,
the
3 The
abbreviation
used
is:
CHO,
Chinese
hamster
ovary.
CANCER RESEARCHVOL. 37
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Polyamines and DNA Synthesis after Heat Shock
per ml, 100 units penicillin, 0.25 @g
Fungizane, and 100 @g
streptomycin. Cultures were maintained in exponential
growth at 37°in humidified 5% C02-95% aimincubators. Cell
doubling times were 13 to 15 hr under these conditions. Cell
numbers were determined using an electronic particle
counter (Caulter Electronics, Hialeah, Fla.). Cells were syn
chronized by the selective detachment of cells in mitosis
(28) which were then cold-accumulated to obtain a large
number of cells at the beginning of each experiment (29).
These cells (mitatic index greater than 85%) were then
plated into T-25 flasks (Falcon Plastics, Oxnard, Calif.) con
taming medium at 37°and allowed to progress through the
cell cycle. In cultures used to measure intra- and extracellu
lamamounts of polyamines, the medium contained 10@ M
aminoguanidine bicarbonate (Sigma Chemical Co. , St.
Louis, Mo.) to inhibit palyamine degradation by diamine
oxidase (25) present in the serum. This treatment did not
alter intracellular palyamine amounts or culture doubling
time for at least 48 hr after addition of aminoguanidine.
Cultures were heated to 43°in the T-25 flasks by immem
sian of the sealed flasks in a temperature-controlled water
bath (temperature constant to ±0.1°)
as previously de
scnibed (6). In all experiments, cells were harvested from
these flasks by scraping with a rubber policeman.
Measurement of DNA Replication Kinetics. This prace
dure has been described in detail in previous reports (8, 15).
Prior to synchronization, cultures were grown for 20 to 24 hr
in medium containing [‘4C]thymidine,0.15 @.&i/ml(53 mCi/
mmole; Schwartz/Mann, Orangebung, N. V.). After synchra
nization and hyperthenmic treatments, the culture medium
was adjusted to 50 @g/mlfan 5-bnamadeaxyumidineand 0.1
pg/mI for 5-fluomodeaxyumidine. Sample plates were then
harvested at various times after treatment with the cell
pellets stared at —15°
in buffer containing 0.15 M NaCI:0.01
M EDTA:0.01
M Tnis, pH 8. Depnateinized
DNA was obtained
by extraction in chlomofonm:isaamyl alcohol (24:1). The per
centage of DNA replicating semicanservatively at any time
after treatment was determined by measuring the pnopon
tion of [‘4C]DNA
that acquired hybrid density in CsCl gra
dients [4.0 ml of 62.8% (w/w) CsCI in 0.01 M Tnis at pH 8.1
and 0.7 ml of deproteinized DNA per sample]. Centnifuga
tian was for 45 hr in a SW 50.1 rotor at 33,000 rpm (20°)in a
preparative ultnacentmifuge. Density information was ob
tamed by measuring the refractive indices of samples, and
radioactivity was assayed on a SeamleMark II liquid scintilla
tian spectrometer.
Determination of PolyamIne Concentrations. This
method has also been previously outlined in detail (14, 21).
Briefly, intracellular palyamine levels were determined as
follows. Cells (5 x 106/sample) were disrupted by sonica
tion with an E/MC Corporation ultrasonic cell disnuptom
(4.5-inch probe) in 400 @l
of 10% tnichboroacetic acid at 0.2°.
The homogenates were centrifuged at 10@x g for 15 mm. A
75-pi abiquot was then placed an a Dumrum Model D-500
automatic amino acid analyzer far assessment of putmes
cine, spermidine, and spermine concentrations (14, 21).
To assay far extracellulan polyamines, 3 ml of medium
were washed with 2 ml of cold 10% tnichbanoaceticacid and
vortexed. Samples were centrifuged at 20,000 rpm in a
Sarvall 55-34 motor for 15 mm. The resulting pellet was
resuspended in 1 ml of 10% tnichlaroacetic acid and centmi
fuged as before, and the supemnatants from bath spins were
combined. Following 2 washes of these supemnatants with
15 ml of anhydrous ether, an equal volume of 12 N HCI was
added. This solution was then sealed in an extraction tube,
hydrolyzed far 16 hr at 110°,and then dried to powder form.
Samples were redissolved in 200 @l
of 0.1 N HCI with 75-id
aliquots assayed as stated before. [14C]Spenmidine was
used to monitor recovery rates.
RESULTS
DNA ReplIcation Kinetics following Hyperthermlc Expo
sures during G1 or S Phase. Exposure of exponentially
gnawing CHO cells to 43°heat for 1 hr reduces single-cell
survival (as measured by colony-farming ability) to appnoxi
mately 20% under the conditions reported here (see Ref. 30
for complete hyperthermic survival data fan this cell line).
While 80% of the cells exposed to this heat shock may
ultimately die, Chart 1 demonstrates that exposure at 43°
either during G1(3 to 4 hr after mitosis) an S phase (9 to 10 hr
after mitosis) does not result in immediate cell lysis, since
theme is no appreciable decrease in cell numbers in the
treated cultures compared to controls. In both instances,
cell division is inhibited. By 20 to 25 hr after mitasis, a small
number of treated cells have begun to divide, in contrast to
cells gnawing normally at 37°,which have completed 1
division by 16 hr after mitosis. Cell numbers in the treated
cultures continue to increase after 20 to 25 hn, finally reach
ing twice the original number by 48 hr after mitasis. During
this time, from 20 to 50 hr after mitosis, the cell number
increase in the treated cultures probably reflects both lysis
of dead an dying cells as well as division of viable cells.
Chart 2 shows the [‘4C]DNA
profiles after centnifugation
an CsCI gradients which are used to determine amounts of
DNA replicated. Normal-density DNA bands around Fraction
19 (density 1.70 g/mI), while newly replicated DNA, which
includes 5-bromodeoxyunidine in place of thymidine, bands
around Fraction 12 (density 1.75 g/ml). Chart 2, A and B,
shows DNA distributions from untreated cultures that were
harvested at 12 and 24 hr after mitasis, respectively. Chart
a)
E
.0
z
a)
02
a)
>
a)
cc 1
0
5
ib
15
20
25
30 45
50
Hoursafter Mitosis
Chart 1. Relative cell number increase for normally progressing control
cells (C) and cultures exposed to 43°heat in mig-G1 phase at 3 to 4 hr after
mitosis ( 0) or in S phase at 9 to 10 hr after mitosis (0). Values represent the
average of 3 separate determinations.
FEBRUARY1977
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483
E. W. Gerner and D. H. Russell
2, C and 0 shows similar data from cultures exposed from 9
to 10 hr after mitosis to 43°heat and then harvested
at 12
and 24 hr, respectively. As seen in Chart 1, theme is only
minimal cell lysis or cell division occurring at these times in
the treated cultures. It is apparent at both times (comparing
Chart 2A with Chart 2C and Chart 2B with Chart 2D) that the
43°-treated cultures show a lower proportion of their
[14C]DNAin the hybrid-density or newly replicated region of
the gradient as compared to controls. This indicates a
suppression of DNA synthesis.
This heat-shock-induced suppression of bulk DNA syn
thesis is described in more detail in Charts 3 and 4. Chart 3
shows the kinetics of semiconservative DNA replication fan
control cultures and for cells exposed to 43°during G1at 3
to 4 hr after mitosis. Control cultures begin to show measun
able newly replicated DNA by 6 hr after mitosis and have
completed 1 round of replication by 18 hr after mitasis. In
contrast, cultures exposed during G1 to 43°heat have nat
replicated measurable amounts of DNA until 9 hr after mito
sis and have replicated less than 75% of their DNA by 25 hr
after mitosis. While the mateof DNA replication is initially
reduced after treatment of these G1 cultures, near-normal
synthetic rates are recovered by 15 to 25 hr. During this time
(Chart 1), cell lysis appears to be minimal, so that this
recovery of DNA replication rates is not an artifact of im
pending cell death.
The results in Chart 4 describe the effects of heat expo
sure at 43°,9 to 10 hr after mitosis, which is during S phase
of the CHO cell cycle, on DNA replication kinetics. By 10 hr
after mitosis, approximately 15 to 20% of the population's
DNA has already replicated (see Charts 2 and 3). Since 5lOr
@- .
A
@0
a)
C)
a)
z
a
)
5
10
15
20
25
30
35
40
45
50
Hours after Mitosis
Chart 3. DNA replication kinetics for synchronized cells treated in mid-G
phase(3to 4 hr after mitosis)at 43°.
•,
controls. 0, valuesfrom treatedcul
tures. 5-Bromodeoxyuridine and 5-fluorodeoxyuridine were added to all cul
tures at 4 hr after mitosis.
@0
a)
Do
C.)
a)
cc
z
a
B
Hours after Mitosis
Chart 4. Semiconservative DNA replication kinetics for synchronized cells
treated in S phase (9 to 10 hr after mitosis) at 43°.•,
controls; 0, values from
cultures exposed to 43°heat. 5-Bromodeoxyuridine and 5-fluorodeoxyuri
0.5
dine were added to the cultures
“z
at 10 hr after mitosis.
0
x
u.u
D
0.
0
::——
0
10
20
300
10
20
30
Fraction Number
Chart 2. [‘4c]DNA
distributions in CsCI gradients. Fraction numbers in
crease from the bottom to the top of the centrifuge tubes. The 1st 40 drops
from the bottom
of each tube were discarded,
and all subsequent
samples
contained 9 drops. The DNA banding around Fraction 12 has a density of 1.75
g/mI and corresponds
to 5-bromodeoxyuridine-substituted
or hybrid
DNA
(15).The DNAbanding around Fraction 19 has a densityof 1.70 g/ml and
corresponds to DNA not containing
5-bromodeoxyuridine
(unreplicated
DNA).A andB, DNAdistributionsfrom untreatedcultures 12and 24 hr after
mitosis
(5-bromodeoxyuridine
and 5-fluorodeoxyuridine
added at 10 hr). C
and0, distributionsfrom culturestreatedat 43°
(at9 to 10hr after mitosis)12
and 24 hr, respectively,after mitosis.The percentageof DNAreplicatedis
then determined by adding the total counts appearing in the hybrid region
and then dividing by the total counts in both the hybrid and normaldensity
DNA regions.
484
bnomodeaxyumidine and 5-fluomodeoxyunidine were not
added to either control on 43°-treatedcultures until after the
exposure to 43°heat, this amount of DNA replication will not
be measured in these experiments. This explains why maxi
mum percentage of DNA replication attained in the control
cultures is only 80% as determined by CsCI gradient analy
sis. Similar to the results for cells treated in mid-G1, the mate
of semiconservative DNA replication initially was reduced
after exposure at 43°.In contrast, however, the mate of
replication did not recover to control matesand remained
reduced in these S-phase-treated cultures through 45 hr
after mitosis (35 hr after the heat shock). Also, the final
percentage of DNA replicated was only 12.5% lower in the
43°-treatedculture (67% at 45 hm)than in the control culture
(79.5% at 24 hr). This indicated that cultures exposed to 43°
heat in S phase are able to complete nearly a full moundof
replication.
Pobyamlne Levels in Synchronized Cells after 43°Expo
sure in G1or S Phase. Chart 5 shows the intracellular levels
of the 3 polyamines, putrescine (Chart 5A), spermidine
(Chart 5B), and spenmine (Chart 5C), at different times after
CANCER
RESEARCH
VOL. 37
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synchronization in mitasis. The closed symbols represent
values from control cultures progressing normally through
the cell cycle at 37°,while the open symbols illustrate polya
mine levels for cells exposed to 43°heat during G1from 3 to
4 hr after mitasis. Intracellular levels of all 3 polyamines
decrease as cell division occurs. Intracellular spemmidine
and spermine amounts then meaccumulate nearly 2-fold by
10 to 12 hr after mitasis as these CHO cells progress
through S phase. When these synchronized cells are ex
posed in mid-G1 phase to 43°heat, an immediate depletion
of intracellular spermidine and spemmine levels is observed,
while putrescine amounts seem unaffected. When the 43°treated cultures are returned to 37°at 4 hr, spermidine and
spermine levels begin to increase and reach control values
by approximately 15 hr after mitasis. Here the results in
Chart 3 should be noted. The maximum mateof DNA replica
tian in control cultures is attained between 8 and 10 hr after
mitosis and by 15 hr after mitasis in cultures exposed to 43°
heat in G1phase. This closely corresponds to the attainment
of high levels of spenmidine, especially as shown in Chart 5
far bath controls and cultures treated during G1at 43°.
In order to determine the fate of the intracellular polya
mines that are lost following heat exposure at 43°and to
determine whether the increases in spermidine and sperm
me observed in control and treated cultures are due to new
synthesis or simply transport from outside the cell, the
extracellubammedium was assayed fan polyamines. Tables 1
to 3 summarize intra- and extracellubar amounts of putres
cine, spermidine, and spermine, respectively, at various
times after mitosis for control cultures and cultures ex
posed to 43°heat in G1(3 to 4 hr after mitosis) and S (9 to 10
hr after mitosis) phase. These tables also express total
culture levels (intra- plus extracellulan levels) and give the
ratio of intracellular to extracellulam amounts. The intracel
lular data (except for cultures treated from 9 to 10 hr) are
taken from Chart 5, and all values are expressed as nmoles/
106 cells. Table 1 shows little variation in putmescine
amounts, either as a function of cell cycle position or after
heat exposure at 43°with the exception of S-phase-treated
cultures. In these cells, themeis an increase in intracellular
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485
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CANCERRESEARCHVOL. 37
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Polyamines and DNA Synthesis after Heat Shock
@
putmescine from approximately 0.5 nmole/106 cells deter
mined immediately after 43°exposure to amounts greaten
than 1 nmale/106 during the following 10 hr (through 20 hr
after mitosis). During this same time themeis a decrease in
extracellular putrescine. The major variation in total culture
putmescine occurs in the S-phase-treated cells where a de
crease in total putrescine is observed at 20 hr (10 hr after
exposure at 43°).Here also, an increase in the ratio of
intracellular to extracellulan putrescine is observed. For all
values listed, this ratio is an the order of 0.15 to 0.3 except
in the cultures exposed to 43°heat at 9 to 10 hm,whene the
ratio increases from 0.24 at 10 hm,to 0.79 at 15 hr and finally
to 1.30 at 20 hr after mitasis.
In contrast to the minor changes observed in putrescine,
spemmidine levels undergo large changes, both as a func
tion of cell cycle phase and in response to heat shock. Table
2 lists the intra- and extraceliulan amounts of spermidine,
showing the near doubling in intracellular levels as cells
move through the division cycle, while extracellulam values
remain essentially unchanged. Note here that while there
may be small fluctuations in extracellulan levels of spenmi
dine during the cell cycle, the variations are statistically
questionable and certainly smaller than the statistically sig
nificant increase in intracellular values that occurs between
8 and 10 hr after mitosis. When cultures are exposed to 43°
heat during G1amS phase, intracellular levels are decreased
50% am more with a coordinate increase in extmacelbulan
amounts. Table 2 also demonstrates that, while intra- and
extracellulam levels of spemmidine change following heat
shack, total culture values remain similar through 8 hr after
mitosis. In control cultures, a significant increase in total
culture spermidine is observed at 10 hr, representing new
synthesis of spenmidine in the culture. Cultures exposed to
43°heat in G1 phase show an increase in total culture
spemmidine beginning at 12 to 15 hr after mitasis. Cultures
treated during S phase at 43°do not show any major in
crease in total spermidine through 20 hr after mitasis. In
contrast to putmescine, which showed intracellular to extra
cellular ratios of 0.15 to 0.30, spenmidine ratios are greater
than 1 at all times for control cultures. After heat exposure
at 43°either in G1an S phase, this ratio decreases as low as
0.12. Combined with the observation that total spemmidine
levels do not change (for example, see 4 hr after mito
sis), this means a lass of intracellular spemmidine with an
increase in extraceblulam spemmidine. Near normal S-phase
ratios are attained by 15 to 20 hr after mitosis fan G1-phase
treated cultures, while S-phase-treated cultures have not
attained control values even by 20 hr after mitasis.
Table 3 shows intracellular and extnacellulan spermine
data. Similar to spermidine, although not as marked, 43°
exposure causes a decrease in intracellular amounts of this
molecule with a subsequent increase in extraceblular bevels.
Intracellular spermine levels seem to accumulate to near
normal S-phase levels following 43°exposure either in G1or
S phase.
It should be noted here that cell numbers for these cub
tunes were always determined after the hyperthenmic treat
ment when samples were harvested at the times shown in
the charts and tables. This fact plus the observation (Chant
1) that appreciable cell lysis does not occur through 20 to 25
hr after mitasis in either G- or S-phase-treated cultures sug
gests that the observations reported here are not simply an
artifact of lysing cells.
DISCUSSION
The data reported here fan synchronous cultures pro
ceeding through the cell cycle at 37°confirm previous find
ings that intracellular palyamine concentrations increase as
cells leave G1 phase and progress through S and G2phases
(11, 23). This relationship is conserved in cultures exposed
to 43°heat in G1 phase. An immediate result of this treat
ment is a depletion of normal intracellular spermidine and
spemmine amounts, followed by a delay in DNA replication
kinetics. As spermidine and spermine reaccumulate, DNA
replication proceeds at a reduced mateuntil near normal 5phase levels of these polyamines are attained. At this time,
the rate of DNA replication increases far a time to appraxi
mately that of control cultures. These results are in agree
ment with those of others, who showed that heat shock
inhibited DNA synthesis (16, 18) and that some cells treated
during G1were abbeto enter S phase but were then blacked
in that cell cycle compartment
(12).
An important implication of our results is that 43°expo
sure in either G1or S phase causes an immediate change in
the transport properties of the cytoplasmic membrane
which leaves it porous to the outward flow of spenmidine
and spermine into the extraceblular medium. This conclu
sian is in agreement with the results of others who have also
observed hyperthermic treatment-induced changes in cell
membranes (1, 9).
Our data are also of interest in comparison with the work
of others who have studied the effects of heat shack on RNA
and DNA synthesis. Intracellular spermidine and spenmine
levels seem to parallel RNA and DNA accumulation (3, 4, 19,
24) leading to the postulate that polyamine biosynthesis is
involved in the control of both RNA and DNA synthesis.
Thus the inhibition of RNA synthesis (26) and DNA synthesis
(16, 18) caused by exposure to high temperatures may be
due to the depletion of normal intracellular pobyamine con
centratians. Our results show that, as spermine and spenmi
dine levels reaccumulate to normal values after 43°expo
sure in mid-Gi, DNA replication proceeds at a near-normal
rate for a time. In contrast, when cells are exposed to 43°
heat during S phase, spermidine levels are reduced and do
not neaccumulate oven the next 10 hr. In these cells, DNA
replication proceeds but at a much reduced rate compared
to controls.
In terms of the effects of 43°exposure for 1 hr on cell
kinetics, our data suggest that cells heated in mid-G1 (3 to 4
hr after mitosis) are inhibited from entering S phase (i.e.,
replicating their DNA) for a few hr and are then able to
replicate DNA, but at a reduced rate for a period of time
before attaining normal rates. in these mid-G1-tneated cub
tunes, only 75 to 80% of the DNA is replicated by 50 hr after
mitosis, while control cultures have replicated 1 moundby 18
to 20 hr. Since cell division is mecommencing by 25 hr after
mitosis in the 43°-treatedcultures and about 20% of the
cells will remain viable after this treatment, it seems likely
that while all the cells are initially inhibited from replicating
FEBRUARY1977
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487
E. W. Gerner and D. H. Russel!
their DNA, same cells are able to complete at least 1 full
round of DNA replication during this time. Since nearly a full
mound of DNA replication occurs after 43°exposure in S
phase, our results would suggest that hyperthemmia causes
a large extension of the time S-phase cells will remain in S
phase, in agreement with the conclusions of others using
different techniques (12).
When comparing the ratio of intracellular to extracellular
levels of each of the 3 polyamines, putmescine ratios are an
the order of 0.2 for control cultures. Spemmine ratios are
nearly 1, and spermidine ratios are even higher, with values
ranging from 1 to 1.5. This must indicate specific cell mem
brane transport mechanisms fan each polyamine. Putres
cine, with its low intnacellulam-to-extnacellulan ratio, seems
not to be affected by exposure to thermal stress (except at
later times after S-phase cultures are exposed to heat
shack), while both spemmidine and spermine, which have
higher ratios, are greatly affected. The observation that the
intracellular accumulation of high bevelsof spermidine and
spenmine, in particular, is temporally related to maximum
bulk DNA-synthetic rates suggests a possible causal reba
tionship between these 2 molecular events. Results to be
published elsewhere show that the polyamine-biosynthetic
enzymes, omnithine decarbaxylase and the S-adenosylme
thionine decanboxylases, are also temporally related to bulk
DNA-synthetic rates in a manner similar to that of intraceblu
lamspermidine and spermine levels shown here. The fact
that the ratio of intracellular to extracellulam spermidine and
spermine must meappnaachcontrols fan DNA replication to
attain maximdm rates further suggests that the transport of
these molecules across cell membranes, as well as their
biosynthesis, may be involved in the DNA synthetic process.
That these 2 processes, biosynthesis and transport across
cell membranes, both may be involved in DNA synthesis is
further suggested by the results in Table 2 which show that
extracellular spemmidine levels may begin to increase be
tween 6 and 8 hr after mitasis, while intracellular levels
increase between 8 and 10 hr after mitosis in control cub
tunes. These increases occur along with maximum S-adeno
sylmethionine decamboxylase activity being expressed at
about 6 hr after mitasis (unpublished results). Evidence that
polyamine accumulation is necessary fan optimal DNA syn
thesis has recently been reported by others using pmace
dunes different from those reported here (5). In this regard,
it is necessary to consider
the results of exposing
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research.
The Relationship between Polyamine Accumulation and DNA
Replication in Synchronized Chinese Hamster Ovary Cells after
Heat Shock
Eugene W. Gerner and Diane Haddock Russell
Cancer Res 1977;37:482-489.
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