Hydration, volume changes and nuclear magnetic resonance proton

relaxation times of HeLa S-3 cells in M-phase and the subsequent cell

cycle

DENYS N. WHEATLEY1, MARGET S. INGLIS1, MARGARET A. FOSTER2

and J. EWEN RIMINGTON2

'Department of Pathology and zBiomedical-l'hysics and Bio-linginecnmi, L'niversity Medical Buildings, Foresteiiiill, Aberdeen . IW 2/1),Scotland, t K

Summary

Most mitotic HeLa cells divided into twodaughter cells with half the volume of the parent;no additional reduction in volume was detectableduring late telophase or the early part of Gx.Synchronous growth throughout the next gener-ation cycle was exponential, 'without an ad-ditional and sudden rise in volume being detect-able as cells entered M-phase. Since the wetweight/dry weight ratio and the protein per unitmass of cells remained constant throughout thecycle, measurements based on volume changesaccurately reflected growth parameters and cellwater content.

Nuclear magnetic resonance (n.m.r.) t\ (spin-lattice) proton relaxation times for intracellularwater for M-phase cells measured at both 32 MHz(25°C) and 80MHz (37°C) in a Bruker spec-trometer had the same values as normal inter-phase cells. Furthermore, cells arrested inmetaphase by nitrous oxide or alkaloids gavethe same f, values. Artificial manipulation of cell

hydration by adjustment of the tonicity of theexternal medium led to t{ values that correlatedwell with the level of intracellular water. On thisbasis, a 40% increase in hydration raised t\values by a factor of 1-29 at 32 MHz and 1-5 at80MHz. This is considerably less than the in-crease by a factor of 1-9 in t\ time reported withthe 40% rise in cell water accompanying mitosis,measured at 30 MHz and 25 °C under isotonicconditions by others. The protein-synthesizingability of hydrated cells was reduced to only halfthe normal level after a doubling of intracellularwater.

The data from these several different analyses,taken together, strongly indicate that the watercontent of mitotic cells is very similar to that ofinterphase cells, and that certain features uniqueto the mitotic phase of the cell cycle cannot beascribed to an elevated (free) water content.

Key words: HeLa, n.m.r. spectrometry, hydration, cellcvcle, mitosis, division.

Introduction

It has been reported that the t\ nuclear magneticresonance (n.m.r.) time of mitotic (iW-phase) HeLacells is increased by as much as a factor of 1 -9 relative tothat of interphase cells (Beall el al. 1976). The periodof elevation was almost exclusively restricted to M-phase itself, and the inference of a large increase in cellwater was confirmed from wet weight/dry weightanalysis, which indicated a 40% rise during mitosis.More recently, Ngo et al. (1984), Ngo & Woolley(1986) and Belfi et al. (1986) have also reported

Journal of Cell Science 88, 13-23 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

substantially longer (by a factor of 1-9-20X) /,relaxation times for Chinese hamster V79 and mouseC3H10 1/2 cells in .U-phase.

Changes as dramatic as a 40% rise in cell watercontent might be considered readily visible by lightmicroscopy, but this is not necessarily the case. In aspherical cell of, say, 16 ^m diameter, a 40% increasein cell volume represents no more than a 9 % increase indiameter. Also the rounding-up of cells going intomitosis and their spreading on return to interphasemakes it difficult to appreciate volume changes. How-ever, a sudden and sizeable increase in hydration

13

associated specifically with mitotic division couldneatly explain a number of characteristics of mitoticcells, e.g. rounding-up and detachment from thesubstratum, increased size of amino acid pools (Mac-millan & Wheatley, 1981), increased thermosensitivity(Rao & Engelberg, 1965), and depressed protein syn-thesis (Fan & Penman, 1970). In addition, the notionthat increased hydration might be required for success-ful passage of cells through M-phase is attractivebecause it offers an explanation for the finding thatprogression can be prevented by the withdrawal ofwater from mitotic cells in hypertonic media (Stubble-field & Mueller, 1960; Wheatley & Angus, 1973), aprocedure that subsequently led to the development ofa reversible method of synchronizing cells in division(Wheatley, 1974).

Our preliminary studies indicated that t\ and tzproton relaxation times increased predictably in HeLacells progressively swollen in a range of hypotonicmedia. On this basis, however, a 40 % rise in cell watercontent increased the t\ time by a factor of only about1-25-1-30, whereas in studies referred to above ahigher factor of 1-9 was found. We have thereforeinvestigated the relationships between cell volume,water content and n.m.r. relaxation parameters moreclosely for mitotic and non-mitotic (interphase) HeLacells, and in cells passing synchronously through orarrested at specific stages of the cell cycle.

Materials and methods

Cell culturing

HeLa S-3 cultures were grown in Eagle's Basal medium(BME) in monolayer or a modified suspension version(Mueller et al. 1962). Their generation time averaged 22h.Mitotic collections were made in many experiments by theshake-off method of Robbins & Marcus (1964) withoutrecourse to presynchronization or mitostatic agents.Synchronized populations with >95 % mitotic cells wereincubated as suspension cultures for n.m.r. sampling duringthe subsequent cell cycle. Where mitotic arrest was required,colcemid at l -5xl0~7M was used or, alternatively, exposureto N2O at 5-7 atmospheres for 3 or 6h before shake-off (Rao,1968) with resuspension in fresh medium to avoid theparamagnetic effects of this gas (Ling, 1983), but normallywithout the presvnchrony treatments used by Beall's group.

To synchronize cells in different cycle phases, hydroxyureaat 2XlO~JM or thymidine at 5xlO~3M was added for 16h tosuspension cultures before the cells were allowed to enter 5-phase synchronously by resuspension in fresh medium.

Cell volume measurement

A Coulter FN' counter coupled to a C-1000 channelyser wasused (Coulter Electronics Ltd, Luton, England). For con-firmation, cell diameters were measured at right angleswith a carefully calibrated Vickers' image-splitting eyepiece(Vickers' Instruments, York, England).

Hydration

Wet/dry weight ratios were obtained from pellets of 2X107

cells spun out at 3000if for 2 mm and subsequently dried toconstant weight over a period of 4 days in a 60-70cC oven.

Protein

Protein was estimated by the modified Folin-Ciocalteaumethod of Oyama & Eagle (1956).

n.m.r. spectrometry

This involved a model CXP100 machine (Bruker, Rheinstat-ten, West Germany) with a temperature control unit oper-ating at either 25 or 37(± 0'5)°C. Twenty points were used tomeasure t\, each point being the mean of four determi-nations. Tau extended to l x / | (1-25). A least-squares fitweighted to remove the skewing effect of the semi-log plot onthe errors was used to determine /;. Examination of theresiduals showed them to be random and a ̂ goodness-of-fittest showed the data to be monoexponential (P>99-9%). tiwas measured by a Carr-Purcell-Meiboom-Gill sequence.Most measurements were made at the more sensitive fieldstrength of 80 MHz, but for comparative reasons 32 MHz wasalso used. Pellets usually of 1X107 to 2X107 cells werecentrifuged at 3000^ for 2 min at 37°C, wicked on the surfaceafter draining and the sides of the n.m.r. tubes were driedwith medical-wipe tissue before stoppering. Measurement of/ ] , ti and proton density were made within minutes there-after. Procedures other than this simple pelleting of sus-pended cells in normal medium will be described whereappropriate in Results.

Radioactive labelling

L-[4,5-3H]leucine at 1850 GBq minor ' (50Cimmor ' ) wasobtained from Amersham International (Amersham, Bucks,England). The culture medium containing 3X 10~4M-leucinewas supplemented with 3 7 K B q m r ' (1 ̂ iCiml"1) of tritiatedleucine to achieve labelling of protein, the radioactive leucinenot significantly altering total leucine concentration.Measurement of incorporation was made on samples of 5 mlfrom treated and control cultures taken at 30 and 60min,cooled rapidly in 5 ml ice-cold saline containing 10~zM-leucine. Samples were routinely washed and prepared forscintillation counting and protein estimation as described (seeWheatley, 1982), to give a specific activity of incorporatedleucine per fig cell protein.

Results

Coulter techniques

Fig. 1 and Table 1 show volume changes of cellsmoving through mitosis after a normal shake-off collec-tion at to- Fig. 2 gives the appearances of cells over60 min after shake-off, with many anaphases havingdeveloped by t2o- Most cells were in mid-late telophaseby /40 and the vast majority reached early G\ by t^.Physical separation of daughter cells by disruption oftheir mid-bodies did not occur for approximately afurther 2h in many cells, particularly when maintained

14 D. N. Wieatlw et al.

in suspension culture, but they were easily detached bygentle shearing forces, i.e. expressing samples througha pipette five times. The failure of separation in'unsheared' samples led to the broadening of the M-phase peak with a shift towards the lower channels of

I50 100/0

Channel number

50 100

Fig. 1. Coulter channelyser size distribution curves with'o~'3Mi referring to min of incubation after collection ofcells in mitosis (thick lines). The panels on the left to <60min indicate the size reduction accompanying mitosis insamples sheared to create a monodispersion of cells, whichwould otherwise remain mostly as doublets, giving abroader peak shown in the /|2o sample on the top of theright panel. The right panel follows the fate of cells leftunsheared throughout the 6h following collection. Thethin line in the /40 panel indicates original position ofmitotic cells before doublets formed; and in the /|2n panel,it indicates the position that (sheared) G'I cells had reachedat /H ).

part of the population (Fig. 1), doublets giving a signalsize some 10% less than a single sphere of equivalentvolume. This was consistent with evidence of alteredelectronic sizing characteristics of adherent latexspheres of equal volumes (R. W. Lines, CoulterElectronics Ltd, personal communication). On separ-ation following shearing, daughter cells were exactlyhalf the volume of the parental mitotic cell (Fig. 1 andTable 1).

By the next (less synchronous) division at =22 h, noevidence was found of a sudden or significant rise ofabout 30-40% in cell volume associated with late Gzcells entering iW-phase as reported by Beall et al. (1976;and see also Fig. 3).

Image-splitting eyepiece

This approach permitted direct measurements on indi-vidual cells. Cells within the modal range for mitoticHeLa of 2500-4200 fl were analysed in and just afterM-phase (see Table 2). The data established thatanaphases and metaphases had identical volumes, i.e.elongation of the cell was accompanied by an appropri-ate reduction in radius at the equator. Daughter cells atlate telophase averaged half the volume of their pro-genitor metaphases. It also confirmed that the sizingcomplication associated with doublets in late ana-phase-early telophase was an artefact of the Coulterelectronic (impedance) method of estimation of theoriginal Coulter system.

More importantly, cells moving into and through theearly hours of G\ never fell below half the jl/-phasevolume. About 65 % of divisions were equal within theaccuracy of the measurements (=5%), 27% wereapproximately 'equal' within twice this error limit, but8% were decidedly unequal, with daughters averagingcell volumes of 1250 fl and 1650 fl (equal division gave1430-1450 fl).

Wet weight/dry weight and wet weight/proteinratios at mid G\ (6h), mid 5 (14h), late S/early Gz(20 h) and GzJM (22 h) are reported in Table 3. Theformer ratio was consistently 5-20 ± 0-08 (i.e. 83-84%water), and the latter was equally consistent at aprotein concentration of 11-45 ± 0-55 f.ig per 100fig wet

Table 1. Electronic (Coulter) cell-sizing of M-phase to early G/ HeLa S-3 cells

collection

12345

Mean volumes

• Doublets prec

% Metaphases

9192979396

( t lS .D. )

lominate at lw when

% Mitosis

9595989898

late telophase

Meta

31433172323133563299

3240 ± 8i

cells remain

Average cell volumes

Doublets*

302029%299131333048

8 3038 ± 58

joined together. At tM

' «

15901560159516371607

1598 ±27

onwards, thev

(fl, urn3)

' i 2 n

16381609164016931663

1649 ±31

were separated bv

'2411

1722—

16651778—

1722 ±57

pipetting.

Hydration of M-phase HeLa cells 15

V

B ftFig. 2. Photomicrographs of normal mitotic HeLa S-3 cells at: A, /(), shake-off collection; B, tw, mostly doublets; andC, /«), after shearing to separate last telophase-early G'I cells. X523; stain, crystal violet.

Table 2. Direct estimation of volumes of M-phase to early G/ cells, calculated fmm diameters measured with a\'icker's image-splitting evepiece

Mctaphase Anaphasc ' 1Z<>

Number cells measuredMean cell volume (fl) ± 1 S.D.

1062874 ±336

1002827 + 280

2111418 ± 177

1011471 ±236

Hours into cell cycle

Fig. 3. Volume change in HeLa cells throughout a cellcycle from D to D. The points show the mean cell volume,and the upper and lower limits of the size distributioncurve are plotted to enclose the stippled area embracing thewhole cell population. The curve is exponential (seeWheatley, 1986).

weight (Table 3). Both ratios were highly comparableto those for asynchronous suspensions, normal mitoticcells and mitotic cells collected by blocking proceduresfor 5-6 h before shake-off (Table 3).

n.m.r. relaxation measurementsCells were pelleted at 37°C and measured withinminutes at the appropriate temperature. Previous work(unpublished) has shown that the conditions chosen(3000g'for 2min) cause little detectable perturbation ofmetabolic processes, though higher speeds and longertimes do produce short interruptions of, e.g., proteinsynthesis. To ensure that cells were not deterioratingrapidly during measurement, n.m.r. measurementswere performed on pellets repeatedly over severalhours. No significant alteration of /) times was detecteduntil 2h after collection, at which time t\ started todecrease, probably due to dehydration effects. Cellscould be easily resuspended by gentle pipetting afterpelleting, with only a small loss in cell number, andwere capable of growing after a short delay, even whenmaintained as pellets for up to 8 h at 23°C.

This early stability, coupled with the completelynormal appearance of pelleted cells in electron mi-croscopy (Fig. 4) led to the selection of cell pelletsrather than suspensions for these experiments. This

16 D. X. Wheat lev et al.

choice eliminated the need to add such extraneoussubstances as relaxants, whose effect on live cells isfrequently injurious and whose use negates the aims ofthis study.

M-phase versus interphase cells. In each exper-iment, we attempted to compare mitotic cells with bothinterphase cells grown under the same condition and anasynchronous control culture, which could be prepared

for n.m.r. measurements with minimal manipulation.The data we obtained are not easy to collate with thefindings of others on account of both the diversity ofcell lines used and the conditions under which relax-ation times were obtained.

For 'unperturbed' mitotic collections, i.e. shake-offcollections not involving prior synchronization, t\ andtz relaxation times at 37°C and 80MHz, and at 15°C

Table 3. Weight/dry weight ratios, water and protein content of HeLa S-3 cell pellets collected by differentmethods for n.m.r. analysis

A.

B.

C.

D.

E.

• Not signi

Culture sample (treatment)

Asynchronous suspension: (1)

(2)(3)

Interphase monolayer: (trypsinized)(scraped)

Mitotic (simple shake-off)(colcenud 3-5 h before shake-off)(N2O 5-5 h before shake-off)

Synchronous (shake-off)Mid-SLatc-i'/earlv 6'2G2/MMid C,

Medium strength adjustment (%; 100% is normalasynchronous culture at isotonicitv)

' 60100ISO200300

ficant; -f" significant, A* < 0-05; J for absolute cell-water

Wet/dry \vt ±S.D.

5-270 ±0-085-473 ±0-195-09810-05

5-280±0-124-509 ±0-35

5-394± 0175-494 ±0-095-470 ±0-11

5-389 ± 0 1 55-570 ±0-225-268 ±0-145-591 ±0-27

7-4235-2413-9273-2682-563

contents, see Fig. 5.

Relative %intracellular water

84-5084-5583-60

84-0881-85

84-3684-6084-54

84-2984-7884-0084-09

88-13184-0279-9776-5771-93

% Protein incell mass ± S.D.

11-45 ±0-551204 ±0-1812-13 ±0-21

11-97 ± 014f*14-11 ±0-42f

l l-96±0-21*12-04 ±0-1811-97 ±0-40

11-78 ±0-2511-33 ± 0-511201 ±0-3011-32 ±0-05

8-4611-8715-5919-2524-27

Fig. 4. Electron micrograph of pellet of HeLa S-3 cells centrifuged at: A, 300; B, 1000; and C, 3000 |f for 2min (seeTable 6). Arrows in C indicate the very small amount of interstitial space. X2700.

Hy drat ion of M-phase HeLa cells 17

and 32MHz are given in Table 4. No difference wasobserved from interphase cells, and therefore weresorted to the use of alkaloids and N2O to accumulatecells in mitosis. Again, no obvious or consistent differ-ence between the M-phase and their correspondinginterphase cells or asynchronous controls was found(Table 4).

n.m.r. values through the cell cycle. Cells at differ-ent stages of the cell cycle were compared following twosynchronizing procedures. One was to allow unper-turbed M-phase collections of cells to continue to growand be sampled during the next generation; the otherwas to use chemical synchronization to entrain cells atthe G\/S boundary and then release them through acycle, sampling at appropriate intervals (see also Table4B). Irrespective of the frequency, temperature andcell cycle stage chosen for analysis, we failed to find anysignificant or consistent pattern of alteration in t\ timesof synchronized cells over the equivalent of a completegeneration time (Table 4).

Analysis of HeLa cells from different culture con-ditions and by different methods of sample preparation.Some preparative procedures and treatments com-monly led to changes in t\ times (or gave erraticvalues). The most troublesome was the removal ofcells grown in monolayer by scraping techniques (seeTable 5). Although Beall (1982) recommended Teflonscraping, any such technique led to cell disruption andthe collection of firmer pellets by centrifugation, whichtended to give lower t\ values, presumably with the lossof much of the 'soluble' material into the supernatantrather than the pellet. Suspension culturing used forour asynchronous controls and for chemical synchron-ization (Tables 4 and 5) usually gave more consistent t\and t-i values. Removal of cells by simple shake-offresulted in suspensions, which were subsequentlyhandled in the same manner. For monolayers, wefound that the only reliable method of release wasgentle trypsinization (0-15% in Dulbecco's phosphate-buffered saline at pH7-2, 295 mosmolar) for theminimum period of time before restoring medium to

Table 4. Relaxation times for HeLa cells of M-phase and at other cell cycle stages

Field strength/temp.80MHz/37°C 32MHz/25°C

Cell preparationRelaxation times (ms)

' 1 ('2) ' 1

j1/-phase collections:(1) 'Unperturbed'shake-off

Interphase control (gentle trypsin)Asynchronous control

(2) Colccmid 3-5 h collectionInterphase control (scraped)Asynchronous control

(3) N2O (5-5 h) collectionInterphase (gentle trypsin)Asynchronous control

B. Synchronized M cells and their progression through the cell cycle:(1) Unperturbed synchronized M cells

+ 1-5 h (early C,)+ 3-5 h (early G,)+6h (mid G',)+7-5 h (mid C )+ 17h (midS)+ 22-5 h (Cz/M)Asynchronous control

(2) N2O (5-5 h) collection 1442Reversed 2 h (early C,) 1444Reversed 7 h (mid C,) 1499

(3) HU*-induced synchrony16 h arrested C i/S) 1439Reversed 3-5 h (mid S) 1444Reversed 8-5 h (C2/M) 1497Reversed 16 h (mid Ci) —Asynchronous control 1431

145814971454146714451436

144215181454

:yclc:14561487146314691487151914361440

(107)(102)(117)(119)(126)(111)

(129)(126)(119)

(102)(87)———

(107)(92)(92)

862849891882769891———

——

————

(138)—

(118)—

(114)

906821923885891

hHU, hydroxvurea.

18 D. N. Wlieatlev et al.

Table 5. n.m.r. relaxation times of asynchronous, HeLa cell populations collected and measured underdifferent conditions

PreparationsFrequency

(MHz)Temperature (±S.D.)*

M'2

MA. Medium: without serumf

Medium: with 10% neonatal calf serum

B. Asynchronous suspension:

Gentle trypsinization

C. Asynchronous suspension:300£pcllet§

1000#pellet§3 0O0#pellet§7 000^ pellet

10000^ pellet

D. Asynchronous monolayer:

Gentle trypsinization

Rubber policeman

Teflon scraper

*S.D. values are the standard deviations of the slopes of the relaxation curves for individual samples from four separate determinations,•f Pure water gave a t\ of 4700 ms.% Range at 80MHz, 1296— 1590ms (mean ± S.D., 1432d: 67) over entire experimental programme. With more standardized collection and

packing procedures, the range fell to 1383-1509ms (mean±s.D., 1433±36ms). At 32MHz, f, was 848±87ms.§ See Fig. 4.

8080

80323232

8080808080

803280328032

3737

37252525

3737373737

372537253725

3821 (±0-04)3500 (±0-01)

1433 (±6)t848 (±8)t896 (±4)887 (±3)

1590 (±1)1515 (±4)1452 (±4)1462 (±7)1467 (±5)

1398 (±5)885 (±5)1135 (±2)665 (±4)1209 (±3)719 (±5)

32742375

106116129121

————

131—138—118

give a cell suspension (Table 5). This same trypsintreatment given to suspension cells did not alter their tor <2 values.

The intensity of centrifugation had some effects onrelaxation times of the resulting pellets. Because cellskept at 37°C can move quickly through M-phase(Fig. 2), it was important to keep the time of cell-pelletpreparation to a minimum (~5min). Pellets producedat low speeds of around 300 |f for 2 min were relativelyfirmly packed at the base of the centrifuge tube, but thecells were well separated higher up (Fig. 4A). After2min at 1000g firm pellets were seen, but gapscontaining extracellular water were observed mainlydue to the microvilli not flattening (Fig. 4B). At 3000 |jffor 2 min, cells packed well without significant extra-cellular space (Fig. 4C; Table 5). The cells themselveswere incompressible, i.e. they did not exude intracellu-lar water, even when grossly hydrated cells were spunat from 3000 £ to 10000^.

Experimental alteration of cellular hydration. Byintentionally altering cell water content with gradedhypotonic and hypertonic media (Wheatley et al. 1983,1984), the relationship between hydration and t\ relax-ation time for asynchronous HeLa cells spun for 2 minat 3000 £ was followed (Fig. 5). A rise of 40% inhydration increased t\ from 1450 to 1868ms, i.e. by afactor of =1-29. This was smaller than the increase ofl-9x for A/-phase cells with a reported 40% increase in

3000

0 1Relative tonicity of external medium (1 >: 295 mosmolar)

Fig. 5. Relationship between /) relaxation time (ms) ofcells placed in media of varying tonicities and their relativecell water content (cell water content in normal basalEagle's medium taken as unity).

intracellular water (Beall et al. 1976). The data make itmost improbable that our M-phase cells have a signifi-cantly elevated water content as this would have beenreadily and accurately detected by n.m.r. spec-trometry. Increasing tonicity drove out labile cell wateras described by Wheatley et al. (1983) and raised

Hydration of M-phase HeLa cells 19

Table 6. Tonicity, cell volume change and protein synthetic activity in HeLa S-3 cells

Medium strength in niosmol (% of control) 295 (100)

Mean cell volume (fl) (% of control) 2110 (100)

Average^ rate protein synthesis (% of control) 100

236(80) 177(60) 118(40) 59(20)

2572(121-9) 3101(142-2) 4247(201-3) [5022]* [(238-0)]*

80-3 52-7 26-6 [2-3]*

•Values in 20% strength medium arc for intact cells; since some lysis was occurring, the values will be distorted; the values in squarebrackets are therefore less reliable.

t Averaged from four rate estimations over 0-30 and 30-60 mm after exposure of cells to hypotonicity by the incorporation of[•"Hjleucine into 5% perchloric acid-prccipitable cell protein.

protein concentration (see Table 3), and correspond-ingly lowered relaxation times (Fig. 5); t\ and es-pecially <2 curves became distinctly multiexponential.

Protein synthesis and cell hydration. Adjustment ofthe external medium to lower tonicity led to a markedincrease in cell volume through the influx of water andto a reduction in the ability of cells to incorporate['HJleucine into protein. This work was carried outwith asynchronous suspension cultures, and the resultsin Table 6 clearly demonstrate that their water contentmust be more than doubled before protein synthesisfalls to 25-30% of the control rate, i.e. to the level ofsuppression in M-phase cells reported by others (Fan &Penman, 1970; Tarnowska & Baglioni, 1979).

Discussion

While a transient hydration associated with the M-phase of the HeLa cell cycle might neatly explain anumber of characteristics of A/-phase cells, the com-bined evidence presented above can only lead to thefirm conclusion that water content and, on average, thefreedom of intracellular water is the same as in inter-phase cells. Furthermore, there is little evidence of anysignificant change during the cell cycle, unlike thefindings of Beall e/ al. (1976), Ngo etal. (1984), Ngo &Woolley (1986) and Belfi et al. (1986), but more inaccord with those of Muller et al. (1980). We havelooked at other available cell types grown in tissueculture at 80 MHz and 37°C, and/or 32MHz and 25°Cand found small variations (e.g. t\ for BHK21/C13 was1730 ms, and Landschiitz ascites tumour cells 1602 ms,cf. HeLa 1464 ms in same experiment; CHO-10 cells atthe lower field strength and temperature was 864 mswith HeLa under the same conditions being 848ms).Our results clearly establish that cells progressing in asynchronized manner from an unperturbed start in M-phase show a remarkably constant wet weight/dryweight ratio and protein content, confirming the n.m.r.evidence.

Cells arrested in mitosis for up to 6 h with alkaloidsgave / | values similar to those of 'normal' mitoticpopulations (Table 4), as did nocodazole-arrested cells(Muller et al. 1980). These collecting agents destroy

microtubules, unlike N2O, which appears to alter theirfunctional efficiency (Brinkley & Rao, 1973). Theagreement between the relaxation values for mitotic cellarrested by these quite different methods makes itunlikely that microtubules are significantly affectingthe overall proton relaxation times (cf. Beall et al.1976).

The most significant observation indicating that atransient increase of about 40 % in cell water is unlikelyto occur during M-phase comes from the results withhypotonic cell swelling. By expressly increasing cellwater content by this amount (Fig. 4), it has beenshown beyond doubt that significant rises in both t\ andti would have easily been detected. There must,however, be reasons for the discrepancies between ourresults and earlier findings, and some of our additionalobservations (Table 5) may be of assistance in thisrespect. The poorer the condition of cells at collection,i.e. the more debris present (visualized close to theordinate axis of the Coulter counter size-distributionanalyser in Fig. 1), the lower the relaxation times of thepellet. In most experiments cells removed from mono-layers gave a lower value than that of the correspondingshake-off mitotic populations. On the other hand,whenever suspension cultures were used throughout,or run in parallel with shake-off collections, similarrelaxation times were found. When cells had beengrown in monolayer for two or more days instead of12-20 h, scrape-off collections using a soft rubber or aTeflon scraper produced a 'suspension' in which sheetsof flattened and often disrupted cells predominated.This material packed down as a hard pellet with farmore protein than intact cells (Tables 3, 4, 5). How-ever, for stricter comparison we found release ofinterphase with a gentle (minimal) trypsinization waseffective, and this same treatment given to mitotic cellsproduced no change in n.m.r. values. In synchronyexperiments carried out entirely with suspension cul-tures, cells at different stages of the cell cycle gavesimilar relaxation times, and this value agreed with thatfor asynchronous culture. The lack of an appropriate,strict control in monolayer experiments may be partlythe source of error. This could be mimicked bysubjecting an asynchronous suspension culture to one

20 D. N. Wlieatlw et al.

pass of a Dounce homogenizer before pelleting. Break-ing of cells in this way was comparable on Coulteranalysis to scraped monolayer preparations and gavelower relaxation times and higher protein content.Nevertheless, these findings could not account for theresults of Belfi et al. (1986), who used an elutriator tofractionate MCA-TCL-15 murine cells into differentcycle phases with regard to volume. Thus the problemis not yet satisfactorily resolved.

Samples of less than ~ 2 x 106 cells often gave low anderratic relaxation times. We identified two possiblereasons for this: first, a relatively greater proportion offragments and contaminants to cells in the pellets; and,second, a tendency for relaxation times to fall whilebeing measured, probably due to relatively large waterloss by evaporation from the sample into the 4-5 mlvolume of warmed air in the sealed tube over it. Pelletsof 2x 107 cells have given us the most consistent t\ and<2 values. Despite the fact that these cells can beresuspended and grown with only a minimal initial lossin cell number (even after as long as 8 h in a stopperedn.m.r. tube at 23°C), n.m.r. measurements mustinevitably be made under less than 'normal physiologi-cal' conditions. All previous investigations on mam-malian cells have met the same problem and criticism,but with our preparative procedures we have attemptedto reduce some of the time delays, temperature shiftsand extracellular space complications, that could havejeopardized the validity of some of the previous com-parative studies. Until an n.m.r. microscope is avail-able to make measurements on normal, in situ, unper-turbed cells, we will have to resort to the crowding ofcells into pellets.

Although we have concentrated on t\ values, we havenoted that, in general, l-i relaxation times changed inregister with the former, ti curves were often multi-exponential. As anticipated (see Fig. 5), cells thathad had a considerable amount of intracellular waterremoved by hypertonic treatment, gave the clearestdemonstration of multiexponential t2 curves. Intra-cellular water exists in different compartments withdifferent degrees of structuredness, and therefore whenthe more labile water is removed, a greater percentageof highly structured water associated with 'insoluble'cellular matrix remains (Wheatley et al. 1984; Clegg,1984).

The data presented in Tables 4 and 5 and throughoutthis study, while showing that the average watercontent is unchanged during mitosis, do not precludethe possibility that changes may occur in the compart-mentalization of water during mitosis. The values for t\and t-i relaxation times are only averages for pellets oflxlO7 to 2xlO7 cells and one cannot resolve matterswith any greater confidence at the present time. In-deed, one would suspect that proton relaxation timeswithin the individual compartments of the cell would

be changed. Aguayo et al. (1986) have shown that thenucleus in a single cell (albeit a Xenopus oocyte) hasmuch more free water than the cytoplasm; duringmitosis, this water must be redistributed as the nuclearcompartment breaks down. If the nuclear water inHeLa cells, with their greater nuclear/cytoplasmicratio, also has this greater degree of freedom, as mightbe expected from the results of Ranade et al. (1979) inmammalian tissue, then this would lead to considerablewater restructuring when a cell goes through mitosis.

Division of HeLa S-3 cells yields daughters of halfthe volume of the 'parent' mitotic cell on most oc-casions, with no obvious further reduction in volume asthey leave M and enter G\. There is an exponential risein volume to the next division, with no sudden surge of- 3 0 - 4 0 % in late G2 as cells pass into M (Fig. 3).These findings, coupled with the constancy of thewet/dry weight ratio and the protein content per unitmass through the cycle, are relevant to the problem ofthe growth kinetics of mammalian cells through the cellcycle (see Prescott, 1976, for a review). This alsoconfirms the detailed analysis of cell growth kinetics(Wheatley, 1986), which addressed the problem ofprotein turnover during growth. Since synthesis ofprotein during mitosis is said to be depressed (Fan& Penman, 1970; Tarnowska & Baglioni, 1979),increased hydration might be responsible for thisdiminution. It is clear from Table 5 that an increase incell water of no less than 100% is required to depressprotein synthetic activity to 25—30% of its interphaselevel. Although suppression does occur with increasinghydration, the lack of any evidence that hydration risesin M-phase indicates that suppression of protein syn-thesis during mitosis must be due to some entirelydifferent mechanism.

Finally, we should not ignore the finding of Wolff &Pertoft (1972) that mitotic cells have a lower buoyantdensity than interphase cells. Mitotic cells fractionatedon a polyvinylpyrrolidone-Eagle's minimal essentialmedium—Ludox colloid silica gradient by centrifu-gation were found between relative densities of 1-041and 1-050, whereas interphase cells were found be-tween 1-050 and 1-058. Although this experiment canbe criticized for employing only a short and low-speedcentrifugation rather than isopycnic banding, the datasuggests that mitotic cells are lighter, than interphases;factors such as cell size and shape producing differentdrag effects might generate some of the apparentdifference. A 40% increase in cell volume in ilV-phasedue to the imbibition of pure water at a density of 1 -000would lower interphase density from an average of1-056 to 1-040, which could account for the reportedchange. Such a change is not reflected in our study by acorresponding fall in protein concentration or wet/dryweight ratio (Table 3). Thus, the problem remainscontentious and more careful investigations arc

Hydration ofM-phase HeLa cells 21

required to establish whether there is a significantuptake, restructuring and re-compartmentalization ofcell water during the mitotic process.

We thank Dr James S. Clegg (Bodega Marine Laboratory,CA, USA) for his criticisms of the manuscript, and Mr R. W.Lines (Coulter Electronics Ltd, Luton, England) for adviceon sizing problems with the Coulter apparatus. This workwas supported in part by grant no. 5600136 from theUniversity of Aberdeen, and by grant no. 813010 from theMedical Research Council for the NMR studies. Technicaland secretarial assistance was kindly provided by Mrs JanetC. Robertson, Miss Isobel Davidson and Miss Ann H.Mackay.

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(Received 23 February J 987-Accepted 27 April 19S7)

Hydration of M-phase HeLa cells 23