The need of living systems for inorganic ions hasbeen documented extensively over the years, butthe reasons for these ion requirements have insome cases remained nebulous. In the case of thetrace elements a number have been shown to operate in specific enzymatic systems. The bulk ions,such as Na+, K+, and C1, enter similarly intoknown enzymatic reactions; however, they alsoappear to have other physiologic functions associated with their osmotic effects and influence onmacromolecular behavior. These latter functionshave not been extensivelystudied in mammalian cellculture systems, but they obviously strongly influence the cell's character and economy. The ionrequirements of mammalian tissue culture cells

a This work was supported by Grant No. C—1897 from theUnited Stat.es Public Health Service and by the Alexanderand Margaret Stewart Trust Fund.

t Predoctoralfellow supportedby the NationalScienceFoundation.

Received for publication July 15, 1960.

were studied by Eagle (8), but his restriction to theparameters of cell increase and visual examinationdid not reveal the striking alterations in metabolism and composition of cells confronted with asodium chloride concentration change, as described in this report.

It should be understood from the outset thatvariation of the sodium chloride concentration inthe medium results in osmotic alteration as well asin changes in Na+ and Cl ion concentrations. Noattempt was made in the following experiments tocompensate for altered osmolarity, and thereforethe results must he interpreted in terms of a combination of osmotic and ionic effects which we asyet have not been able to dissociate.

MATERIALS AND METHODS

Cell culturea.—Thegeneral methods used in theroutine cultivation of strain HeLa in our laboratory have been described elsewhere (17). Glassattached cultures subcultured at weekly intervals

1646

Effects of SodiUm Chloride Concentration on Growth, Biochemical Composition, and Metabolism of HeLa Cells*

ELTON STUBBLEFIELDt AND GERALD C. MUELLER

(McArdle Memorial Laboratory, 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 optimalNaCl concentration for growth and deoxyribonucleic acid (DNA) synthesis was 100—130 mr&.Increase of the culture medium NaCl concentration from 1920m@mto 92920m@mresulted 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 increasingNaCl concentration up to 92920mr@&,as did also the relative conversion of glucose into lactic acid.

Synthesis of DNA decreased concomitantly with cell proliferation. The high-salteffects were reversible by restoration of 1920mr& NaCl concentration in the medium.

High-salt medium transiently increased the phase contrast density of the cellnucleus and caused chromosome clumping in mitotic cells. Although the cells in 2920mM NaC1 medium increased in cell size, there was no marked alteration of the typicalepithelioid appearance of the cells.

The data are discussed in terms of a concept involving nuclear and cytoplasmicphases of a cell life cycle. A possible ionic control mechanism regulating DNA synthesis and initiation of the nuclear cycle is presented.

No attempt was made to dissociate the osmotic effects of NaCl variation fromthose effects due specifically to the Na@ and C1 ions.

Research. on August 20, 2018. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

STUBBLEFIELD AND MUELLER—Effects of NaCI on HeLa Cells @l647

have provided cells for experimentation. Cells weretrypsinized for experiments 3 days after subcultureto assure logarithmic growth.

Foreach experiment 0.5 million cells in 10 ml. ofmedium were aliquoted replicately into 3-oz. pharmacy bottles, overlaid with an atmosphere of S percent CO2 in air, and grown as a monolayer at3170 C. The medium was that formulated by Eagle

(7) containing 10 per cent bovine serum and92X 10@ M inositol, with Earle's balanced salt solution' as the electrolyte base.

Experimental cultures were terminated eitherby trypsinization (for cell counts) or by fixation ins@itu(for biochemical analysis). Cultures washedwith isotonic saline were overlaid with 5.0 ml. of0.05 per cent trypsin in a calcium-magnesium-freeHanks balanced salt solution (15). After 15 mmutes of incubation at 37°C., 5.0 ml. of growth medium containing a natural trypsin inhibitor in theserum was added to inactivate the trypsin, and thedetached cells were dispersed by pipetting. Cultures to be analyzed biochemically were washedconsecutively with 5 ml. of cold 0.9 per cent NaCl,4 per cent perchloric acid, 80 per cent ethanol, absolute ethanol, and ether, and, finally, the cellswere allowed to air dry. The solutions were carefully poured in and out of the culture bottle in sucha way that the cell monolayer was not detached.

Upon termination of a culture, the medium wascentrifuged to remove any suspended cells andthen frozen for later analysis. Any detached cells sorecovered from the medium were enumerated asdead cells, since such cells are largely nonviable incloning experiments and appear dead (i.e., pyknotic nuclei) under visual examination.

Tryp€inized cells were enumerated in a brightline hemocytometer and more recently by electronic gating with a Coulter Counter,2 with equivalent results.

BiOChemicol analy8es.—Cultures fixed 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 removedbydethc*tinnoversodiumhydroxidechipsunderreduced pressure. DNA was measured by thefluorome1@ic analysis of Kissane and Robins (192).Salmon sperm DNA was used as the primarystandard. For RNA, the Ceriotti ribose analysis(6) was employed. The Qyama and Eagle modification (16) of the Lowry analysis for protein wasthe method used for culture protein determinatioii,with bovine serum albumin as a standard. Thecombined sicohol and ether extracts of cultures

1 All salts used in the medium were Baker's reagent grade.

2 Coulter Automatic Blood Cell Counter and Cell Sim

Analyzer. Coi.êter Electronics, Chicago 40, Ill.

were ashed and analyzed for phosphorus by theFiske and Subbarow method (9) to obtain an indexof phospholipide content of the experimental ciiitures.

Two biochemical parameters were followed inthe medium—glucose depletion and lactic acid accumulation. Glucose was measured colorimetrically with glucose oxidase.3 Lactic acid was analyzed according to the method of Barker and Summerson (92), designed for use with blood samples.

Cytologic vwthod.—Cell volume measurementswere made on the trypsinized cell suspensions. Aconcentrated suspension of the spherical cells wasphotographed on a hemocytometer grid. In ananalysis of the photograph, cell diameters could becompared directly with the known dimensions ofthe hemocytometer grid. The average volume offrom 50 to 100 cells was then computed.

For cytologic studies, cultures were grown atcomparable cell densities on small coverslipsplaced in Petri dishes and incubated in sealed jarsgassed with S per cent CO2 in air. Coverslips werethen removed at appropriate intervals for stainingor phase contrast observations of perfusion experiments.

For perfusion studies, a coverslip (11 X 9292mm.) with the cells attached to one surface wassealed to a clean microscope slide with a beeswaxparaffin mixture (1 : 1) to make a chamber about 1mm. deep containing the cells. All manipulationswere carried out in a high-humidity room at370 C. under a constant stream of 5 per cent CO2 in

air saturated with water vapor. In experiments involving rapid medium changes, the ends of the perfusion chamber were left open. As the test mediumwas introduced at one end, the old medium was absorbed into clean filter paper at the other end. Forlong-term perfusions, glass capillaries were sealedin place at each end of the chamber, and the cultures were fed for several days with a gravity flowsystem at a rate of about 1 ml/hour. Although noattempt was made to maintain absolute sterility,no contamination was encountered.

RESULTS

EFFECTS OF VARIOUS CONCENTRATIONS

OF NaC1

Eagle (8) reported that, whereas NaCl concentrations between 60 and 150 mu allowed growth ofHeLa cells, maximal growth was obtained near 100IRM NaCl. Chart 1 shows the results of a similar

experiment in our laboratory. The cultures grewfaster and tolerated excess NaCI better, but thetwo experiments essentially agreed.

a Glucostat. Worthington Biochemical Corporation, Freehold, N.J.

Research. on August 20, 2018. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

1648 Cancer Research Vol. 920, December, 1960

In this experiment the cells were planted inundialyzed serum medium, incubated for 1 day,and the medium was removed from the attachedcells. Immediately, dialyzed serum media containing various concentrations of NaC1 were introduced. For 3 days cultures in each NaCl concentration series were harvested periodically for cellcounts and biochemical analyses.

(00

xCt)-J-JLUU

CHART 1.—Proliferation of HeLa cells in media containingvarious concentrations of NaCl. At “zerotime,― replicate cultures were fed Eagle's medium containing the indicated NaCIconcentration and supplemented with 10 per cent dialyzed beefserum. The seru@ahad been dialyzed against 0.9 per cent NaC1,and the salt contribution from this source was included in thecalculation of the final NaC1 concentration.

Cell num.ber8.—Several features of the population graph (Chart 1) are noteworthy. The optimalNaCl concentration for growth appeared to be100—iSO mi&, in agreement with Eagle's observa

tion. At concentrations between 70 and 190 mr@i,positive proliferation was obtained, whereas atvery low (40 m3&)or high (9250mimi)NaCl concentrations cell degeneration occurred. Salt concentrations of 70 and 160 m@ did not affect proliferation before 6 hours, and from 6 to 923hours cell increase was retarded. Subsequently, the cells inthese two salt concentrations appeared to recover

and resume growth. Thus, salt concentrationsslightly deviant from optimal did not affect celldivision immediately but appeared to block aprocess of interphase at least 6 hours before mitosis. At extreme hypo- and hypertonicity (40 and190 m@i and above) mitosis itself was interferedwith and cell increase immediately blocked. Above160 m@ NaCl there was an initial loss of cells fromthe monolayer. These cells were dead when recovered from the medium. The generation time was 9292hours under optimal growth conditions (100 and130 mM), whereas the culture grown in 92920m@iNaCl exhibited an apparent population stability,since no change in cell number was seen during theexperimental period.

Cell compositions.—The biochemical compositions of the cells after 3 days in the experimentalmedia are depicted in Chart 92.Whereas RNA andprotein increases appeared in the high-salt culturesand the amount of phosphate derived from thecombustion of lipide material was likewise higherin the 190- and 92920-m@iNaCl cultures, the DNAcontent did not vary appreciably. This suggeststhat the ionic block of proliferation was associatedwith a disproportionate synthesis of cytoplasmiccomponents and an interference with the onset ofthe nuclear reproductive cycle.

Glucosemetabolism.—Theeffect of salt variationon glucose utilization rates after 92days of cultureis shown in Chart 3. The rate of utilization per mg.of protein can be seen to increase with NaCl concentration up to 92920mMNaCl. At NaCl concentrations above 92920mM glucose utilization was inhibited.

The amount of glucose that could be accountedfor as lactic acid also increased with NaCl concentration. Chart 4 is a graph of the ratios of lacticacid production rates to glucose utilization rates,showing this effect on the 92dday.

EFFECTS OF 92920mM NaCl

An increase of the sodium chloride concentration in Eagle's medium from 1920m@ to 92920mr@iresulted itt slowly growing, metabolically alteredHeLa cells. The cell composition of these “highsalt―cultures was acutely altered, with 92-to 3-foldincreases in RNA, protein, and lipide phosphate.DNA content per cell was, however, unchanged.The following experiments describe in detail theculture of HeLa cells under high-salt conditionsover longer periods of time.

Cell number8.—Chart S is a graph of culturegrowth in media of normal (1920 mimi) and high(92920mM) NaC1 content. Curve A shows a 3-daygrowth curve beginning with 0.5 million cells.When on the 92dday the medium was changed to

000000(Y)(Q c'Ju@-@ c'jc@J

MILLIMOLAR NaCI

Research. on August 20, 2018. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

I I I I III

-U

8C)

>

i4OO@@@-U

I3OO@P1

I2OO@

P1

0PhI-r

0 00000N 0(f@OO@(@J

MILLIMOLAR NodCHART 4.—The 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 glucosedepletion rate divided by the lactic acid accumulation rate isthe ratio plotted. A value of 1.00 corresponds to total conversion of glucose to lactic acid.

@.1Z

@., -@4

LU

ICt)Q@

<0

OuJ

o@a@-J

0 0 000000lit N

MILLIMOLAR NciCICHART 3.—The effect of NaCI variation on the rate of glu

cone depletion by HeLa cells after 48 hours of culture. The glucase utilization rate was calculated by dividing the slope of theglucose depletion curve at 48 hours by the quantity of cell protein accumulated in the culture. The results are expressed as,hg. glucose utilized per hour per mg. cell protein.

CHART 2.—Biochemical compositions of HeLa cells grownfor S days in Eagle's medium containing the indicated NaClconcentration. These data were taken from replicate cultures

Ui

@250-

LU -a:

I—-J:DU

ELOO@4%

a:Is@

E

having the corresponding culture populations shown in the 70-hour curve of Chart 1. One picogram equals 1O@ gm.

1.00

0.75-0

!@0.50a:

0.25

.

.

I I I I

100 190

MILLIMOLAR NciCI

Research. on August 20, 2018. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

1650 Cancer Research Vol. 920, December, 1960

one containing 92920mM NaCI and dialyzed serum,there were an initial loss of cells and a subsequentgrowth decrease as seen in Curve B. Curve C represents the total number of cells in the culture, i.e.,living cells attached to the glass surface plus deadcells that could be suspended in the medium. It canbe seen that there was a gradual increase in thenumber of dead cells released from the cell monolayers for the first 92days in high-salt medium.Initially the apparent@tability of the culture population was therefore a dynamic situation in whichcells were proliferating and dying at approximately the same rate. After 92days these processes decreased, and both cell death and proliferation dedined as a less dynamic equilibrium was reached.

Cells die after 4 days in high-salt medium if thecultures are not fed. The last points of Curves Band C illustrate this point in that only 15 per centof the cells were still attached to the glass. Analysis of the medium at this point revealed total depletion of the glucose content; presumably othermetabolites may also have been exhau8ted@ Thisdemonstrates high metabolic activity in these cultures, whereas net growth was minimal.

If the cells were fed more high-salt medium, andrefed after 4 more days, the monolayer populationdecreased more gradually over the next week, asshown in Curve D. The gradual loss of attachedcells was balanced by their appearance in the medium as dead cells (Curve E).

Cultures refed medium containing 1920 mr@iNaCl after 4 days in high-salt medium resumedproliferation 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 ofthe high-salt cultures. Normal growing cultures(Curve A) lose only about 1 per cent of the monolayer into the detached phase per day, whereas, incontrast, cultures treated with 92920mr@iNaCl lose10—15 per cent per day.

Revised culture methods.—A revision ofthe previous experiment is shown in Chart 6 in which allcultures were fed on alternate days throughout theexperiment in orderto prevent medium depletion.Glycine and serine (1 X 10@ M each) were alsoadded to the dialyzed serum, high-salt medium,since Lockart and Eagle had demonstrated a marginal requirement for these amino acids (14). Under these conditions the. cells grew slightly fasterthe first 3 days in high-salt medium, and theymaintained a more constantmonolayer populationthan. previously. More recent experiments withundialyzed serum media . used throughout haveshown that dialysis of the serum was not important.

Cell compositioms.—After several feedings the

monolayer population varied slightly from flask toflask, probably because of a variable loss of viablecells dislodged during the medium changes. In order to circumvent minor fluctuations in composition data due to this lack of exact replication, theprotein and RNA measurements were comparedwith the DNA values from the same flask. A duplicate DNA analysis was then run on an aliquot ofthe trypsinized cell suspension used for cell counts.It was observed that, whereas trypsinization lowered the protein and RNA content of the cells, theDNA was unaffected. The cell suspensions werewashed consecutively with cold solutions of 0.9 percent NaC1, 4 per cent perchloric acid, 80 per centethanol, absolute ethanol, and ether, and air-dried,in a manner analogous to the in situ treatment ofthe cell monolayers.

Analysis of the trypsinized cells revealed arather high level of DNA per cell (Chart 7) at thebeginning of the experiment (924 picograms),which fell within 92@days to the value usually observed (18 picograms). This phenomenon has beenobserved occasionally in association with the mitial growth lag of cultures, but it is not well understood.

The amount of DNA per cell was not appreciably altered by the increased NaCl concentration.Cytologic observations of living perfused cultures@revealed an initial high-salt inhibition of metaphase and a preferential killing of post-divisiondaughter cells ; these processes are reflected in theinitial increase in DNA per cell observed duringthe first 16 hours in high-salt medium. Then, as themetaphase-blocked cells struggled through the division process on the 92dday in high-salt medium,@the DNA per cell decreased to 15 picograms percell and then gradually rose to the normal level of18 picograms per cell.

In contrast, the ratios of protein (Chart 8) andRNA (Chart 9) to DNA climbed rapidly, after aninitial lag, when the cells were exposed to high-saltmedium. Upon reaching levels about twice thoseof the control cultures, the rates of protein andRNA synthesis decreased. Cell volumes are shownin Chart 10, and, in agreement with protein in--crease, the cell volume approximately doubledover the first 4 days in 92920mrti NaCl medium.

Return of high-salt cultures to 1920mr@iNaClmedium resulted in a decrease in RNA, protein,and cell volume after. a 92-day lag. The growingcells assumed their original biochemical composi-.tion.

Cytology.—Cytologic examination of perfusedcultures revealed a transient alteration of nuclear@phase contrast density when the cells were initiallysubjected to high-salt medium. Delineation of nu-

Research. on August 20, 2018. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

I 234567891Ô 1112

TIME IN DAYSCHART 6.—Improved “stabilization― of HeLa cell popula- vent medium depletion, and the Eagle's medium was supple

tions. The experimental design is the same as that shown in mented with glycine and serine (1 X 10@ as each). Curves AChart 5 except that cultures were fed on alternate days to pre- and C—120 mss; Curve B—220 mss NaC1 medium.

(00xC,)

-JUiU

CHART 5.—―Stabiization― of HeLa cell populations by theaddition of NaC1 to Eagle's medium. Curve A shows a 3-daygrowth curve initiated with 0.5 X 10@cells per replicate culture. On day 2 the medium was changed to one containing 220mM NaQ (arrow). Curve B depicts the monolayer populationattached to the glass bottle, and Curve C represents the totalcell population, i.e., living attached cells plus dead cells that

could be suspended in the medium by gentle agitation of theculture. Curves D and E demonstrate the effect of subsequentfeedings of 220 mM NaC1 medium (arrows) to prevent nutrientdepletion (compare with the terminal points on Curves B andC). Curves F and Gdepict the eventualrenewed growth of ciiitures fed normal (120 mM) NaC1 medium.

I

5.0- /AI

4O I— I

3.0- /

. I

II

o2.0- IQ çV@'@ fI

x(1@ f t t.fB

@I0-@ 0.8 E (FEEDING)

0.6-

0.4-

0.3-

I I I0

TIME IN DAYS

Research. on August 20, 2018. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

C,)-J-JLiiUz0-J-J

a:Liia.

zaEa,

CHART 7.—DNA content of normal and “high-salt― HeLa varied only slightly in the 220 rims NaC1 cultures (0). Controlcells. DNA analyses were made on the trypsinized cell suspen- cultures in 120 mas NaCl meduum—(•). Arrows indicate mesions used for cell counts. The amount of DNA per million cells dium changes.

za

4DAYS

50

.@,@/:::T@c40—

z

@20-Ii0 t (FEEDING)

@I0-

a: I I0 1 2 3 4 5 6 7

TIME iN DAYSCLIART 8.—Ratio of protein to DNA in normal and “high

salt― HeLa cultures. Biochemical analyses were made on auquota of the same culture in each case. (•) = 120 @nMNaC1;(0) = @om@ NaCl; arrows indicate medium changes.

3TIME IN

Research. on August 20, 2018. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

STUBBLEFIELD AND MUELLER—Effects of NaC1 on HeLa Cells 1653

clear structure became very difficult, but a normalappearance was restored after several hours.Clumping of metaphase chromosomes, a resultpreviously observed by Hughes (10), was alsoseen. This may be the major cause of the metaphase block that ensued. After 924hours in highsalt medium, 15 per cent of the cells were in metaphase. At 50 hours very few mitoses were observed, so most of the blocked cells must havecompleted division. The monolayer population iscorrespondingly increased. The cells seem to adaptto high-salt conditions, because what appear to benormal mitoses take place after 3 days in 92920m@NaCl medium. The mitotic rate is offset by anequivalent death rate which maintains a constantmonolayer population.

Cultures perfused continuously maintain a normal cytologic appearance in high-salt medium forextended periods of time. One such culture hasbeen observed continuously for 4 weeks. Althoughcell size is increased, most cells maintain a typicalepithelioid morphology.

DISCUSSION

As early as 1911 attempts were made to evaluate the effects of tonicity on tissue cultures (5, 13).Acrudeestimationofexplantsizerevealedoptimalion requirements roughly similar to those acceptedtoday. At that early date Carrel and Burrows (5)conceived of the possibility that ionic balancemight hold normal body tissues in check, preventing or allowing growth as required. Although wenow know that other factors, such as hormones,are active in this role, the ionic influences may stillbe more fundamental. In spite of the homeostaticcharacter of extracellular fluids, the intracellularionic environment may fluctuate over wide ranges,depending on the metabolic state of the cell. Theseionic variations could be mediated by hormonalinfluence on ionic transport systems.

In the absence of hormonal variation, theseionic influences should be demonstrable by variation of the ionic character of the extracellular fluid.This is confirmed by the experiments herein reported. The cells tolerate a wide range of NaClconcentration, but above and beyond this they exhibit an adjustment of composition and metabolism in response to each particular NaCl concentration. Not only do high-salt conditions result inincreased amounts of cellular protein, but preliminary enzyme studies4 indicate radical shifts inthe enzymatic composition of the cell, with a sudden disappearance of some enzymes normallyfound in growing HeLa cells. Presumably the total

4 Unpublished data.

protein increase reflects a corresponding increasein other enzyme species which we have not studied. The increased glucose consumption per unit ofcellular protein supports this conclusion.

Therefore, the over-all effect of an increase inenvironmental NaCl concentration from 1920 to92920m@ approximates a cellular metabolic shiftfrom hyperplasia to hypertrophy. Rapid cell proliferation is halted, and an increase in cell size ensues. The transition is characterized by an acutephase with qualitative changes in cytologic appearance and by the more chronic alterations ofcell composition which follow. The process is reversible by restoration of the lower NaCl level, butthis again requires a significant period of time forreadjustment.

The increased RNA and protein contents of thehigh-salt cultures demonstrate the dissociation ofthese polymer syntheses from cell proliferation.On the other hand, the relatively constant level ofDNA per cell in various NaC1 concentrations reveals a close linkage between DNA synthesis andcell division. If the cell life history can be dividedinto two phases, growth and cell division, then theprimary effect of high-salt medium appears to bein blocking the cell reproduction cycle at a pointantecedent to DNA synthesis and cell division.The first phase might be termed the cytoplasmiccycle, and the second phase the nuclear divisioncycle. Under such a concept, normal differentiatedcells would remain predominantly in the cytoplasmic cycle, e.g., secretory cycle, whereas proliferating cells would combine the cytoplasmic andnuclear division cycles. The process of differentiation and endocrine control could emphasize specifically one or the other cycle in a particular tissue.

Just why high salt concentration interferes withDNA synthesis is not clear, but an interesting possibility is at hand. Itoh and Schwartz (11) demonstrated a relatively higher nuclear sodium contentcompared with that of the cytoplasm in several tissues. This suggests a system transporting Na fromthe cytoplasm into the nucleus. The physical stateof DNA-protein has been shown (3) to be sensitiveto salt concentration, and Bollum (4) found thatDNA polymerase will not work in NaCl concentrations above 100 m@i. In a bacterial system,Bardos et a!. (1) observed what appeared to be aspecific block of DNA replication by elevatedionic strength in the medium. Upon these observations one can build a hypothetical cycle of eventswhereby a cell could regulate its DNA synthesis.During most of a cell life cycle, when DNA synthesis is not occurring, a transport system maintains a

Research. on August 20, 2018. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

a

za:U-0

0

@0.5a:

1.0

t (FEEDING)

I I I I I I I

0 I 2 3 4 5 6 7TIME INDAYS

CHART9.—Ratioof RNA to DNA in normal and “high- strate the dissociation of protein and RNA syntheses fromsalt―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

CHART 10.—Average cell volumes of normal and “high- computed and the averages determined from 50 to 100 cells ussalt―HeLa cells. Cell diameters were measured onphotographs each case. (@) = 120 mas NaCl; (0) = 220 m@ NaC1; arrowsof trypsunized cells and compared with hemocytometer grid indicate medium changes. Compare with Chart S.dimensions in the same photograph. Cell volumes were then

TIME INDAYS

Research. on August 20, 2018. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

STUBBLEFIELD AND Muii@u@—-Effects of NaCl on HeLa Cells 1655

high nuclear Na concentration. Then at some pointin the cycle, if the Na pump shut off, the Na woulddiffuse into the cytoplasm, and DNA replicationcould proceed with initiation of the nuclear cycle.Maintenance of a high extracellular Na concentration might then easily prevent the nuclear Na content from reaching a level low enough to allow anormal rate of DNA synthesis, if the cytoplasmicNa concentration varies with the extracellular concentration.

Increase of other ions may have similar effectson macromolecular systems, and the studies ofBollum (4) and Bardos et al. (1) demonstrate thissituation. Our studies have been restricted toNaCl, however, since this salt is by far the mostabundant ionic species in tissue fluids and is likelyto be more physiologically important. Nevertheless, it is possible that the Na@ ion fluctuationsoperate to produce the observed effects in competition with other ionic entities. In this connectioncomparative studies with KC1 and certain nonionic materials are contemplated.

ACKNOWLEDGMENTS

The authors wish to acknowledge the capable technicalassistance of Mrs. Eleanor Erikson and Mrs. Kathleen Deighton.

REFERENCES1. BARDOS,T. J.; GORDON,H. L.; and HEENAN,E. F. Ionic

Inhibition of Bacterial Growth. II. Relationship of IonicInhibition to DNA-Protein Biosynthesis. J. Am. Chem.Soc., 77:3115—19, 1955.

2. BARKER, S. B., and StmnsEasoN, W. H. The ColorimetricDetermination of Lactic Acid in Biological Material. J.Biol. Chem., 138:535—54,1941.

3. BERNSTEIN,M. H., and MAZIA,D. The Desoxyribonucleoprotein of Sea Urchin Sperm. II. Properties. Biochirn.et Biophys. acta, 11:59—68, 1953.

4. BOLLUM, F. J. Calf Thymus Polymerase. J. Biol. Chern.,235:2399—@403,1960.

5. CARREL, A., and BURROWS, M. T. On the PhysicochemicalRegulation of the Growth of Tissues. J. Exper. Med.,13:562—70,1911.

6. CERIOTTI, G. Determination of Nucleic Acids in AnimalTissues. J. Biol. Chem., 214:59—70, 1955.

7. EAGLE, H. Nutrition Needs of Mammalian Cells in TissueCulture. Science, 122:501—4, 1955.

8. . The Salt Requirements of Mammalian Cells inTissue Culture. Arch. Biochem. & Biophys., 61:356—66,1956.

9. FISKE, C. H., and SUBBAROW, Y. The Colorinietric Determination of Phosphorus. J. Biol. Chem., 66:375—400,19@5.

10. HUGHES, A. Some Effects of Abnormal Tonicity on Dividing Cells in Chick Tissue Cultures. Quart. J. Micr. Sc.,93:207—19, 1952.

11. ITOH, S., and SCHWARTZ,I. L. Sodium and PotassiumDistribution in Isolated Thymus Nuclei. Am. J. Physiol.,188:490—98,1957.

ice.KISSANE,J. M., and RoBINS,E. The Fluorometric Messurement of Deoxyribonucleic Acid in Animal Tissues withSpecial Reference to the Central Nervous System. J.Biol. Chem., 233: 184—88,1958.

13. LEwis, M. R., and Lswxs, W. H. The Cultivation ofTissues from Chick Embryos in Solutions of NaC1, CaCl2,KCI and NaHCO3. Anat. Eec., 5:277—85,1911.

14. LOCKART,R. Z., and EAGLE,H. Requirements for Growthof Single Human Cells. Science, 129:252—54, 1959.

15. MARCUS, P. I.; CIECITJRA, S. J.; and PUCK, T. T. ClonalGrowth in Vitro of Epithelial Cells from Normal HumanTissues. J. Exper. Med., 104:615-28, 1956.

16. OYAMA, V. I., and EAGLE, H. Measurement of Cell Growthin Tissue Culture with a Phenol Reagent (Folin-Ciocalteau). Proc. Soc. Exper. Biol. & Med., 91:305—7, 1956.

17. RUECKERT, R. R., and MUELLER, G. C. Effect of OxygenTension on HeLa Cell Growth. Cancer Research, 20:944—49,1960.

Research. on August 20, 2018. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

1960;20:1646-1655. Cancer Res   Elton Stubblefield and Gerald C. Mueller  Biochemical Composition, and Metabolism of HeLa CellsEffects of Sodium Chloride Concentration on Growth,

  Updated version

  http://cancerres.aacrjournals.org/content/20/11/1646

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/20/11/1646To request permission to re-use all or part of this article, use this link

Research. on August 20, 2018. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from