Effects of SodiUm Chloride Concentration on Growth, Bio
chemical Composition, and Metabolism of HeLa Cells*
ELTON STUBBLEFIELDt
(McArdle
Memorial
Laboratory,
AND GERALD C. MUELLER
Univertity
of Wiaconsin
Medical School, Madi,on,
Wis.)
SUMMARY
Some effects of variation of the sodium chloride content of the medium on growth,
metabolism, and chemical composition of HeLa cells were determined. The optimal
NaCl concentration for growth and deoxyribonucleic
acid (DNA) synthesis was 100—
130 mr&.Increase of the culture medium NaCl concentration from 1920m@mto 92920m@m
resulted in a decreased growth rate and an increase in content of ribonucleic acid
(RNA), protein, and lipide phosphate per cell. Cell volume was likewise increased.
The rate of glucose utilization per mg. of culture protein increased with increasing
NaCl
concentration
up to 92920mr@&,as did also the relative
conversion
of glucose
in
to lactic acid.
Synthesis of DNA decreased concomitantly
with cell proliferation. The high-salt
effects were reversible by restoration of 1920mr& NaCl concentration
in the medium.
High-salt medium transiently
increased the phase contrast density of the cell
nucleus and caused chromosome clumping in mitotic cells. Although the cells in 2920
mM NaC1 medium
increased
in cell size, there
was no marked
alteration
of the typical
epithelioid appearance of the cells.
The data are discussed in terms of a concept involving nuclear and cytoplasmic
phases of a cell life cycle. A possible ionic control mechanism regulating DNA syn
thesis and initiation of the nuclear cycle is presented.
No attempt was made to dissociate the osmotic effects of NaCl variation from
those effects due specifically to the Na@ and C1 ions.
The need of living systems for inorganic ions has
been documented extensively over the years, but
the reasons for these ion requirements
have in
some cases remained nebulous. In the case of the
trace elements a number have been shown to oper
ate in specific enzymatic systems. The bulk ions,
such as Na+, K+, and C1, enter similarly into
known enzymatic reactions; however, they also
appear to have other physiologic functions associ
ated with their osmotic effects and influence on
macromolecular
behavior.
These
latter
functions
have not been extensivelystudied
in mammalian cell
culture systems, but they obviously strongly influ
ence the cell's character and economy. The ion
requirements
of mammalian
tissue culture cells
a This
United
work was supported
Stat.es Public
Health
by Grant
Service
were studied by Eagle (8), but his restriction to the
parameters of cell increase and visual examination
did not reveal the striking alterations in metabo
lism and composition of cells confronted with a
sodium chloride concentration
change, as de
scribed in this report.
It should be understood from the outset that
variation of the sodium chloride concentration
in
the medium results in osmotic alteration as well as
in changes in Na+ and Cl ion concentrations.
No
attempt was made in the following experiments to
compensate for altered osmolarity, and therefore
the results must he interpreted in terms of a com
bination of osmotic and ionic effects which we as
yet have not been able to dissociate.
No. C—1897 from the
MATERIALS
and by the Alexander
AND METHODS
Cell culturea.—Thegeneral methods used in the
t Predoctoralfellow supportedby the National Science routine cultivation
of strain HeLa in our labora
and Margaret Stewart Trust Fund.
Foundation.
Received for publication July 15, 1960.
tory have been described elsewhere (17). Glass
attached cultures subcultured at weekly intervals
1646
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
STUBBLEFIELD
AND MUELLER—Effects
of NaCI
on HeLa
@l647
Cells
have provided cells for experimentation.
Cells were
trypsinized for experiments 3 days after subculture
to assure logarithmic growth.
Foreach experiment 0.5 million cells in 10 ml. of
medium were aliquoted replicately into 3-oz. phar
macy bottles, overlaid with an atmosphere of S per
cent CO2 in air, and grown as a monolayer at
were ashed and analyzed for phosphorus by the
Fiske and Subbarow method (9) to obtain an index
of phospholipide content of the experimental ciii
tures.
Two biochemical parameters were followed in
the medium—glucose depletion and lactic acid ac
3170 C.
lution' as the electrolyte base.
Experimental
cultures were terminated
either
by trypsinization
(for cell counts) or by fixation in
cally with glucose oxidase.3 Lactic acid was ana
lyzed according to the method of Barker and Sum
merson (92), designed for use with blood samples.
Cytologic vwthod.—Cell volume measurements
were made on the trypsinized cell suspensions. A
concentrated suspension of the spherical cells was
s@itu(for biochemical analysis). Cultures washed
photographed
with isotonic saline were overlaid with 5.0 ml. of
0.05 per cent trypsin in a calcium-magnesium-free
Hanks balanced salt solution (15). After 15 mm
utes of incubation at 37°C., 5.0 ml. of growth me
dium containing a natural trypsin inhibitor in the
serum was added to inactivate the trypsin, and the
detached cells were dispersed by pipetting. Cul
tures to be analyzed biochemically were washed
consecutively with 5 ml. of cold 0.9 per cent NaCl,
4 per cent perchloric acid, 80 per cent ethanol, ab
solute ethanol, and ether, and, finally, the cells
were allowed to air dry. The solutions were care
fully poured in and out of the culture bottle in such
a way that the cell monolayer was not detached.
Upon termination of a culture, the medium was
centrifuged to remove any suspended cells and
then frozen for later analysis. Any detached cells so
recovered from the medium were enumerated as
dead cells, since such cells are largely nonviable in
cloning experiments and appear dead (i.e., pyk
notic nuclei) under visual examination.
Tryp€inized cells were enumerated in a bright
line hemocytometer
and more recently by elec
tronic gating with a Coulter Counter,2 with equiv
alent results.
analysis of the photograph, cell diameters could
compared directly with the known dimensions
the hemocytometer
grid. The average volume
from 50 to 100 cells was then computed.
For cytologic studies, cultures were grown
The
medium
(7) containing
was
formulated
10 per cent
92 X 10@ M inositol,
BiOChemicol
that
by
bovine
with Earle's
serum
balanced
analy8es.—Cultures
Eagle
fixed
and
salt so
and
dried
in situ were dissolved in 88 per cent formic acid,
and aliqiiots were taken for measurement of DNA,
RNA, and protein. The formic acid was removed
bydethc*tinn
oversodium
hydroxide
chipsunder
cumulation.
Glucose
was
measured
colorimetri
on a hemocytometer
comparable
cell
densities
on
grid.
small
In
an
be
of
of
at
coverslips
placed in Petri dishes and incubated in sealed jars
gassed with S per cent CO2 in air. Coverslips were
then removed at appropriate intervals for staining
or phase contrast observations of perfusion experi
ments.
For perfusion studies, a coverslip (11 X 9292
mm.) with the cells attached to one surface was
sealed to a clean microscope slide with a beeswax
paraffin mixture (1 : 1) to make a chamber about 1
mm. deep containing the cells. All manipulations
were carried out in a high-humidity
room at
370
C.
under
a constant
stream
of
5 per
cent
CO2
in
air saturated with water vapor. In experiments in
volving rapid medium changes, the ends of the per
fusion
chamber
were left open. As the test medium
was introduced at one end, the old medium was ab
sorbed into clean filter paper at the other end. For
long-term perfusions, glass capillaries were sealed
in place at each end of the chamber, and the cul
tures were fed for several days with a gravity flow
system at a rate of about 1 ml/hour. Although no
attempt was made to maintain absolute sterility,
no contamination
was encountered.
RESULTS
reduced pressure. DNA was measured by the
EFFECTS
OF
VARIOUS
CONCENTRATIONS
fluorome1@ic analysis of Kissane and Robins (192).
OF
NaC1
Salmon sperm DNA was used as the primary
standard. For RNA, the Ceriotti ribose analysis
Eagle (8) reported that, whereas NaCl concen
(6) was employed. The Qyama and Eagle modifi
trations between 60 and 150 mu allowed growth of
cation (16) of the Lowry analysis for protein was HeLa cells, maximal growth was obtained near 100
the method used for culture protein determinatioii,
IRM NaCl.
Chart
1 shows
the results
of a similar
with bovine serum albumin as a standard. The
experiment in our laboratory. The cultures grew
combined sicohol and ether extracts of cultures
faster and tolerated
excess NaCI better,
but the
two experiments essentially agreed.
1 All salts used in the medium
were Baker's
reagent
grade.
2 Coulter
Analyzer.
Automatic
Blood
Coi.êter Electronics,
Cell
Counter
Chicago 40, Ill.
and
Cell
Sim
a Glucostat.
hold, N.J.
Worthington
Biochemical
Corporation,
Free
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
Cancer Research
1648
In this experiment
the cells were planted in
undialyzed serum medium, incubated for 1 day,
and the medium was removed from the attached
cells. Immediately, dialyzed serum media contain
ing various concentrations
of NaC1 were intro
duced. For 3 days cultures in each NaCl concen
tration
series
were harvested
counts and biochemical
periodically
for cell
analyses.
and
Vol. 920, December,
resume
slightly
growth.
deviant
from
Thus,
salt
optimal
1960
concentrations
did not affect
cell
division immediately
but appeared to block a
process of interphase at least 6 hours before mito
sis. At extreme
hypo-
and hypertonicity
(40 and
190 m@i and above) mitosis itself was interfered
with and cell increase immediately blocked. Above
160 m@ NaCl there was an initial loss of cells from
the monolayer. These cells were dead when recov
ered from the medium. The generation time was 9292
hours under optimal growth conditions (100 and
130 mM), whereas the culture grown in 92920m@i
NaCl exhibited an apparent population stability,
since no change in cell number was seen during the
experimental period.
Cell compositions.—The biochemical composi
tions of the cells after 3 days in the experimental
media are depicted in Chart 92.Whereas RNA and
protein increases appeared in the high-salt cultures
and the amount of phosphate derived from the
combustion of lipide material was likewise higher
in the 190- and 92920-m@i
NaCl cultures, the DNA
content did not vary appreciably.
This suggests
that the ionic block of proliferation was associated
with a disproportionate
synthesis of cytoplasmic
components and an interference with the onset of
the nuclear reproductive cycle.
(0
0
x
Ct)
-J
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LU
U
Glucosemetabolism.—Theeffect of salt variation
on glucose utilization rates after 92days of culture
is shown in Chart 3. The rate of utilization per mg.
of protein can be seen to increase with NaCl con
centration
00000
0(Y)(Q
c'Ju@
-@
c'jc@J
MILLIMOLAR NaCI
CHART 1.—Proliferation
various concentrations
of HeLa
cells in media
containing
of NaCl. At “zerotime,― replicate
cul
up to 92920mMNaCl.
At NaCl
concen
trations above 92920mM glucose utilization was in
hibited.
The amount of glucose that could be accounted
for as lactic acid also increased with NaCl concen
tration. Chart 4 is a graph of the ratios of lactic
acid production rates to glucose utilization rates,
showing this effect on the 92dday.
tures were fed Eagle's medium containing the indicated NaCI
concentration
and supplemented
calculation
EFFECTS
with 10 per cent dialyzed beef
serum. The seru@ahad been dialyzed against 0.9 per cent NaC1,
and the salt contribution from this source was included in the
of the final NaC1 concentration.
OF 92920mM NaCl
An increase of the sodium chloride concentra
tion in Eagle's medium from 1920m@ to 92920mr@i
resulted
itt slowly
growing,
metabolically
altered
HeLa cells. The cell composition of these “high
salt―cultures was acutely altered, with 92-to 3-fold
increases in RNA, protein, and lipide phosphate.
DNA content per cell was, however, unchanged.
100—iSO mi&, in agreement
with Eagle's
observa
The
following experiments describe in detail the
tion. At concentrations
between 70 and 190 mr@i,
culture
of HeLa cells under high-salt conditions
positive
proliferation
was obtained,
whereas
at
over
longer
periods of time.
very low (40 m3&) or high (9250mimi)NaCl concen
Cell number8.—Chart S is a graph of culture
trations cell degeneration occurred. Salt concen
growth in media of normal (1920 mimi) and high
trations of 70 and 160 m@ did not affect prolifera
(92920mM) NaC1 content. Curve A shows a 3-day
tion before 6 hours, and from 6 to 923hours cell in
crease was retarded. Subsequently,
the cells in growth curve beginning with 0.5 million cells.
When on the 92dday the medium was changed to
these two salt concentrations
appeared to recover
Cell num.ber8.—Several features of the popula
tion graph (Chart 1) are noteworthy. The optimal
NaCl concentration
for growth appeared to be
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
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0
Ph
I-
o@a@
100
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MILLIMOLAR NciCI
CHART 2.—Biochemical
compositions
of HeLa
cells grown
for S days in Eagle's medium containing the indicated NaCl
concentration.
r
190
having the corresponding culture populations shown in the 70hour curve of Chart 1. One picogram equals 1O@ gm.
These data were taken from replicate cultures
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N
I
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I
III
of NaCI
variation
on the rate
of glu
cone depletion by HeLa cells after 48 hours of culture. The glu
case utilization rate was calculated by dividing the slope of the
glucose depletion curve at 48 hours by the quantity of cell pro
tein accumulated
in the culture. The results are expressed as
,hg. glucose utilized per hour per mg. cell protein.
I
0(f@OO@(@J
MILLIMOLAR Nod
CHART 4.—The
effect
I
00000
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MILLIMOLAR NciCI
CHART 3.—The
I
0
effect
of NaCl
variation
on the ratio
of the
glucose depletion rate to the lactic acid accumulation rate.
Each rate was calculated
as the slope of the depletion
(glucose)
or accumulation (lactic acid) curve at 48 hours. The glucose
depletion rate divided by the lactic acid accumulation rate is
the ratio plotted. A value of 1.00 corresponds to total conver
sion of glucose to lactic acid.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
Cancer Research
1650
one containing 92920mM NaCI and dialyzed serum,
there were an initial loss of cells and a subsequent
growth decrease as seen in Curve B. Curve C rep
resents the total number of cells in the culture, i.e.,
living cells attached to the glass surface plus dead
cells that could be suspended in the medium. It can
be seen that there was a gradual increase in the
number of dead cells released from the cell mono
layers for the first 92 days in high-salt medium.
Initially
the apparent@tability
of the culture
pop
ulation was therefore a dynamic situation in which
cells were proliferating and dying at approximate
ly the same rate. After 92days these processes de
creased, and both cell death and proliferation de
dined as a less dynamic equilibrium was reached.
Cells die after
4 days in high-salt
medium
if the
cultures are not fed. The last points of Curves B
and C illustrate this point in that only 15 per cent
of the cells were still attached to the glass. Analy
sis of the medium
at this point
revealed
total
de
pletion of the glucose content; presumably other
metabolites may also have been exhau8ted@ This
demonstrates high metabolic activity in these cul
tures, whereas net growth was minimal.
If the cells were fed more high-salt medium, and
refed after 4 more days, the monolayer population
decreased more gradually over the next week, as
shown in Curve D. The gradual loss of attached
cells was balanced by their appearance in the me
dium as dead cells (Curve E).
Cultures refed medium containing
1920 mr@i
NaCl after
proliferation
4 days in high-salt
medium
resumed
after a 92-day lag period (Curve F).
Dead cells continued to accumulate in the medium
(Curve G) at a rate roughly equivalent to that of
the high-salt cultures. Normal growing cultures
(Curve A) lose only about 1 per cent of the mono
layer into the detached phase per day, whereas, in
contrast, cultures treated with 92920mr@iNaCl lose
10—15 per
cent
per
day.
Revised culture methods.—A revision ofthe pre
vious experiment is shown in Chart 6 in which all
cultures were fed on alternate days throughout the
experiment
in orderto
prevent
medium
depletion.
Glycine and serine (1 X 10@ M each) were also
added to the dialyzed serum, high-salt medium,
since Lockart and Eagle had demonstrated a mar
ginal requirement for these amino acids (14). Un
der these conditions the. cells grew slightly faster
the first 3 days in high-salt medium, and they
maintained a more constantmonolayer
population
than. previously. More recent experiments
with
undialyzed
serum media . used throughout
have
shown that dialysis of the serum was not impor
tant.
Cell compositioms.—After several feedings the
Vol. 920, December,
1960
monolayer population varied slightly from flask to
flask, probably because of a variable loss of viable
cells dislodged during the medium changes. In or
der to circumvent minor fluctuations in composi
tion data due to this lack of exact replication, the
protein and RNA measurements
were compared
with the DNA values from the same flask. A dup
licate DNA
analysis
was then run on an aliquot
of
the trypsinized cell suspension used for cell counts.
It was observed that, whereas trypsinization low
ered the protein and RNA content of the cells, the
DNA was unaffected. The cell suspensions were
washed consecutively with cold solutions of 0.9 per
cent NaC1, 4 per cent perchloric acid, 80 per cent
ethanol, absolute ethanol, and ether, and air-dried,
in a manner analogous to the in situ treatment of
the cell monolayers.
Analysis of the trypsinized
cells revealed a
rather high level of DNA per cell (Chart 7) at the
beginning
of the experiment
(924 picograms),
which fell within 92@
days to the value usually ob
served (18 picograms). This phenomenon has been
observed occasionally in association with the mi
tial growth lag of cultures, but it is not well under
stood.
The amount of DNA per cell was not appreci
ably altered by the increased NaCl concentration.
Cytologic
observations
of living
perfused
cultures@
revealed an initial high-salt inhibition of meta
phase and a preferential killing of post-division
daughter
cells ; these processes are reflected
initial increase in DNA per cell observed
in the
during
the first 16 hours in high-salt medium. Then, as the
metaphase-blocked
cells struggled
through
the di
vision process on the 92dday in high-salt medium,@
the DNA per cell decreased to 15 picograms per
cell and then gradually rose to the normal level of
18 picograms per cell.
In contrast, the ratios of protein (Chart 8) and
RNA (Chart 9) to DNA climbed rapidly, after an
initial lag, when the cells were exposed to high-salt
medium. Upon reaching levels about twice those
of the control cultures, the rates of protein and
RNA synthesis decreased. Cell volumes are shown
in Chart 10, and, in agreement with protein in-crease, the cell volume approximately
doubled
over the first 4 days in 92920mrti NaCl medium.
Return of high-salt cultures to 1920 mr@iNaCl
medium resulted in a decrease in RNA, protein,
and cell volume after. a 92-day lag. The growing
cells assumed their original biochemical composi-.
tion.
Cytology.—Cytologic
examination
of perfused
cultures revealed a transient
alteration
of nuclear@
phase contrast density when the cells were initially
subjected to high-salt medium. Delineation of nu-
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(0
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U
TIME IN DAYS
CHART 5.—―Stabiization―
of HeLa
cell populations
by the
addition of NaC1 to Eagle's medium. Curve A shows a 3-day
growth curve initiated with 0.5 X 10@cells per replicate
ture. On day 2 the medium was changed to one containing
cul
220
could be suspended in the medium by gentle agitation of the
culture. Curves D and E demonstrate
the effect of subsequent
feedings of 220 mM NaC1 medium (arrows) to prevent nutrient
depletion (compare with the terminal points on Curves B and
mM NaQ (arrow). Curve B depicts the monolayer population
C). Curves F and Gdepict the eventualrenewed growth of ciii
attached
tures fed normal
to the glass bottle, and Curve C represents
the total
(120 mM) NaC1 medium.
cell population, i.e., living attached cells plus dead cells that
I
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I
3.0-
II
/
I
I
o2.0Q
I çV@'@
x
(1@
f
@I00.8 E
@
f
I
t
t.fB
(FEEDING)
0.60.40.3I
0
I
I
I
234567891Ô
1112
TIME IN DAYS
CHART 6.—Improved
“stabilization― of HeLa
cell popula-
tions. The experimental
design is the same as that shown in
Chart 5 except that cultures were fed on alternate days to pre-
vent
medium
depletion,
and
the
Eagle's
medium
was supple
mented with glycine and serine (1 X 10@ as each). Curves A
and C—120 mss; Curve B—220 mss NaC1 medium.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
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a
E
a,
3
4
TIME IN DAYS
CHART 7.—DNA
content
of normal
and
“high-salt― HeLa
cells. DNA analyses were made on the trypsinized cell suspensions used for cell counts. The amount of DNA per million cells
varied
only slightly
in the 220 rims NaC1 cultures
(0).
Control
cultures in 120 mas NaCl meduum—(•). Arrows indicate me
dium changes.
50
z
a
40—
z
Ii
.@,@/:::T@c
0
t
@20-
(FEEDING)
@I0a:
I
1
0
I
2
3
4
5
6
7
TIME iN DAYS
CLIART 8.—Ratio
salt― HeLa cultures.
of protein
to DNA
Biochemical
in normal
analyses
and “high
were made on au
quota of the same culture in each case. (•) = 120 @nM
NaC1;
(0) =
@om@ NaCl; arrows indicate medium changes.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
STUBBLEFIELD
AND MUELLER—Effects
clear structure became very difficult, but a normal
appearance
was restored
after several hours.
Clumping of metaphase
chromosomes,
a result
previously
observed
by
Hughes
(10),
was
also
seen. This may be the major cause of the meta
phase block that ensued. After 924hours in high
salt medium, 15 per cent of the cells were in meta
phase. At 50 hours very few mitoses were ob
served, so most of the blocked cells must have
completed division. The monolayer population is
correspondingly increased. The cells seem to adapt
to high-salt conditions, because what appear to be
normal mitoses take place after 3 days in 92920m@
NaCl medium. The mitotic rate is offset by an
equivalent death rate which maintains a constant
monolayer population.
Cultures
perfused
continuously
mal cytologic appearance
extended
periods
of time.
maintain
in high-salt
One
such
a nor
medium for
culture
has
been observed continuously for 4 weeks. Although
cell size is increased, most cells maintain a typical
epithelioid morphology.
DISCUSSION
As early as 1911 attempts were made to evalu
ate the effects of tonicity on tissue cultures (5, 13).
Acrudeestimationofexplantsizerevealedoptimal
ion requirements roughly similar to those accepted
today. At that early date Carrel and Burrows (5)
conceived of the possibility that ionic balance
might hold normal body tissues in check, prevent
ing or allowing growth as required. Although we
now know that other factors, such as hormones,
are active in this role, the ionic influences may still
be more fundamental.
In spite of the homeostatic
character of extracellular fluids, the intracellular
ionic environment
may fluctuate
over wide ranges,
depending on the metabolic state of the cell. These
ionic variations could be mediated by hormonal
influence on ionic transport systems.
In the absence of hormonal variation, these
ionic influences should be demonstrable by varia
tion of the ionic character of the extracellular fluid.
This is confirmed by the experiments herein re
ported. The cells tolerate a wide range of NaCl
concentration,
but above and beyond
this they ex
hibit an adjustment
of composition and metabo
lism in response to each particular NaCl concen
tration. Not only do high-salt conditions result in
increased amounts of cellular protein, but pre
liminary enzyme studies4 indicate radical shifts in
the enzymatic composition of the cell, with a sud
den disappearance
of some enzymes normally
found in growing HeLa cells. Presumably the total
4 Unpublished
data.
of NaC1
on HeLa
1653
Cells
protein increase reflects a corresponding increase
in other enzyme species which we have not stud
ied. The increased glucose consumption per unit of
cellular protein supports this conclusion.
Therefore, the over-all effect of an increase in
environmental
NaCl
concentration
92920m@ approximates
a cellular
from hyperplasia to hypertrophy.
liferation is halted,
sues. The transition
and an increase
is characterized
from
1920 to
metabolic shift
Rapid cell pro
in cell size en
by an acute
phase with qualitative
changes in cytologic ap
pearance and by the more chronic alterations of
cell composition which follow. The process is re
versible by restoration of the lower NaCl level, but
this again requires
readjustment.
a significant
period
of time for
The increased RNA and protein contents
of the
high-salt
cultures demonstrate
the dissociation
of
these polymer
syntheses
from cell proliferation.
On the other hand, the relatively constant level of
DNA per cell in various NaC1 concentrations
re
veals a close linkage
between
DNA
synthesis
and
cell division. If the cell life history can be divided
into two phases, growth and cell division, then the
primary
effect of high-salt
medium
appears
to be
in blocking the cell reproduction cycle at a point
antecedent
to DNA synthesis and cell division.
The first phase might be termed the cytoplasmic
cycle, and the second phase the nuclear division
cycle. Under such a concept, normal differentiated
cells would remain
predominantly
in the cyto
plasmic cycle, e.g., secretory
cycle, whereas pro
liferating cells would combine the cytoplasmic
and
nuclear division cycles. The process of differentia
tion and endocrine
control could emphasize
cifically one or the other cycle in a particular
spe
tis
sue.
Just why high salt concentration
with
interferes
DNA synthesis is not clear, but an interesting
pos
sibility is at hand. Itoh and Schwartz
(11) demon
strated a relatively
higher nuclear sodium content
compared with that of the cytoplasm in several tis
sues. This suggests a system transporting Na from
the cytoplasm into the nucleus. The physical state
of DNA-protein has been shown (3) to be sensitive
to salt concentration,
and Bollum (4) found that
DNA polymerase will not work in NaCl concen
trations
above
100 m@i. In
a bacterial
system,
Bardos et a!. (1) observed what appeared to be a
specific block of DNA replication
by elevated
ionic strength in the medium. Upon these observa
tions
one can build
a hypothetical
cycle of events
whereby a cell could regulate its DNA synthesis.
During most of a cell life cycle, when DNA synthe
sis is not occurring, a transport system maintains a
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
a
za:
U- 1.0
0
t
0
(FEEDING)
@0.5
a:
0
I
I
I
2
I
I
I
3
4
5
TIME IN DAYS
I
I
6
7
CHART9.—Ratioof RNA to DNA in normal and “high- strate the dissociation of protein and RNA syntheses from
salt―ucLa cultures. (@) —120 m@NaC1; (0) = 220 m@ DNA synthesis in HeLa cells fed 220 m@Eagle's medium.
NaC1; arrows indicate medium changes. Charts 8 and 9 demon
TIME INDAYS
CHART
10.—Average
cell
volumes
of normal
and
“high-
salt―HeLa cells. Cell diameters were measured onphotographs
of trypsunized cells and compared with hemocytometer
grid
dimensions in the same photograph.
Cell volumes were then
computed
and the averages
determined
from 50 to 100 cells us
each case. (@) = 120 mas NaCl; (0) = 220 m@ NaC1; arrows
indicate medium changes. Compare with Chart S.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
STUBBLEFIELD
AND Muii@u@—-Effects
of NaCl
on HeLa
1655
Cells
high nuclear Na concentration. Then at some point
in the cycle, if the Na pump shut off, the Na would
diffuse into the cytoplasm, and DNA replication
could proceed with initiation of the nuclear cycle.
Maintenance of a high extracellular Na concentra
tion might then easily prevent the nuclear Na con
tent from reaching a level low enough to allow a
normal rate of DNA synthesis, if the cytoplasmic
3. BERNSTEIN,M. H., and MAZIA,D. The Desoxyribonucleo
Na concentration
7. EAGLE, H. Nutrition Needs of Mammalian Cells in Tissue
Culture. Science, 122:501—4, 1955.
8.
. The Salt Requirements of Mammalian Cells in
Tissue Culture. Arch. Biochem. & Biophys., 61:356—66,
1956.
9. FISKE, C. H., and SUBBAROW, Y. The Colorinietric
De
termination
of Phosphorus.
J. Biol. Chem., 66:375—400,
19@5.
varies with the extracellular
con
centration.
Increase of other ions may have similar effects
on macromolecular
systems, and the studies of
Bollum (4) and Bardos et al. (1) demonstrate this
situation.
Our studies have been restricted to
NaCl, however, since this salt is by far the most
abundant ionic species in tissue fluids and is likely
to be more physiologically important. Neverthe
less, it is possible that the Na@ ion fluctuations
operate to produce the observed effects in competi
tion with other ionic entities. In this connection
comparative
studies
ionic materials
with
KC1 and
certain
non
are contemplated.
ACKNOWLEDGMENTS
of Mrs. Eleanor
Properties.
Biochirn.
4. BOLLUM, F. J. Calf Thymus Polymerase. J. Biol. Chern.,
235:2399—@403,
1960.
5. CARREL, A., and BURROWS, M. T. On the Physicochemical
Regulation
of the Growth of Tissues. J. Exper. Med.,
13:562—70,
1911.
6. CERIOTTI, G. Determination
of Nucleic Acids
Tissues. J. Biol. Chem., 214:59—70, 1955.
10. HUGHES, A. Some
Effects
of Abnormal
in Animal
Tonicity
on Divid
ing Cells in Chick Tissue Cultures. Quart. J. Micr. Sc.,
93:207—19, 1952.
11. ITOH, S., and SCHWARTZ,I. L. Sodium and Potassium
Distribution
in Isolated Thymus Nuclei. Am. J. Physiol.,
188:490—98,
1957.
ice. KISSANE,
J. M., and RoBINS,E. The Fluorometric Mess
urement
of Deoxyribonucleic
Special
Reference
to
the
Acid in Animal Tissues with
Central
Nervous
System.
J.
Biol. Chem., 233: 184—88,
1958.
13. LEwis, M. R., and Lswxs, W. H. The Cultivation of
Tissues from Chick Embryos
The authors wish to acknowledge the capable technical
assistance
protein
of Sea Urchin Sperm.
II.
et Biophys.
acta, 11:59—68, 1953.
Erikson and Mrs. Kathleen
Deigh
ton.
in Solutions
of NaC1, CaCl2,
KCI and NaHCO3. Anat. Eec., 5:277—85,1911.
14. LOCKART,
R. Z., and EAGLE,H. Requirements for Growth
of Single Human Cells. Science, 129:252—54, 1959.
15. MARCUS,
REFERENCES
1. BARDOS,T. J.; GORDON,
H. L.; and HEENAN,E. F. Ionic
Inhibition
of Bacterial
Growth.
II. Relationship
of Ionic
Inhibition to DNA-Protein Biosynthesis. J. Am. Chem.
Soc., 77:3115—19, 1955.
2. BARKER, S. B., and StmnsEasoN,
Determination
of Lactic
Acid
Biol. Chem., 138:535—54,1941.
W. H. The Colorimetric
in Biological
Material.
J.
P. I.; CIECITJRA, S. J.; and
PUCK, T. T. Clonal
Growth in Vitro of Epithelial Cells from Normal
Tissues. J. Exper. Med., 104:615-28, 1956.
16. OYAMA, V. I., and EAGLE, H. Measurement
in Tissue Culture with a Phenol Reagent
Human
of Cell Growth
(Folin-Ciocal
teau). Proc. Soc. Exper. Biol. & Med., 91:305—7, 1956.
17. RUECKERT, R. R., and MUELLER, G. C. Effect of Oxygen
Tension on HeLa Cell Growth. Cancer Research, 20:
944—49,1960.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
Effects of Sodium Chloride Concentration on Growth,
Biochemical Composition, and Metabolism of HeLa Cells
Elton Stubblefield and Gerald C. Mueller
Cancer Res 1960;20:1646-1655.
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