Serial cultivation of ehrlich ascites tumor cells in hypertonic media

Experimental

SERIAL

CULTIVATION

Cell Research 70 (1972) 317-324

OF EHRLICH

ASCITES

TU

IN HYPERTONIC D. 0. SCHACHTSCHABEL

and 6. E. FOLEY

Institut fiir Physiologische Chemie der Philipps-UniversitBt, 3550 Marburg, BRD, and The Children’s Cancer Research Foundation, and Department of Pathology, Harvard Medical School, Boston, Mass. 02115, USA

SUMMARY Sublines of hyperdiploid Ehrlich ascites tumor cells (ED-O.15 cells) growing in Eagle basal medium (total salt concentration: 0.15 M) supplemented with 10 % fetal calf serum were adapted, by stepwise addition of NaCl to the medium, to growth in hypertonic media at salt concentrations of 0.25, 0.30, 0.35, 0.40, 0.45, and 0.50 M. Progressive increases in the NaCl concentration resulted in a progressive increase in the population doubling time of these high-salt-tolerant cultures. Cells growing in isotonic control medium were predominantly ‘epithelial-like’ in contrast to the flat, extended, ‘fibroblast-like’ morphology of cells growing in hypertonic media. These alterations in cell morphology and growth pattern are interpreted as ‘adaptive’ changes in response to ionic changes in the substrate, since ‘re-adaptation’ to growth in isotonic media resulted in a reversion to the morphology and population doubling time characteristic of the original parent cells. It is suggested that such high-salt-tolerant cells may be useful as model systems for study of the effects of substrate alteration on cellular metabolism.

The effects of variations in tonicity and the individual salts contained in the culture medium on the growth of cells and tissues were investigated more than fifty years ago by Carrel1 & Burrows [3], Lewis & Lewis 1231,Loeb & Fleisher [26] and Ebeling [12]. These early studies were undertaken “to determine what physico-chemical factors could activate the rate of growth” [3]; and “to analyze the factors which determine mitotic proliferation in tissues growing in cuhure media under experimentally varied conditions” [26]. The salt composition of these early media was patterned after the ionic composition of the blood [25], (for review cf [47]); and the bulk cations found to be essential for the survival and long-term growth of mammalian cells in culture were Na+, Kf, Mg2+, and Ca2+ [9, 43, 551. 21.-

711802

A concentration of 85-115 mM Na+ and l-10 mM K+ was required for o~t~rna~growth of mouse fibroblasts and HeLa cells [9]: and significant inhibition of growth was induced by NaCl in concentrations of 150-175 m [9]. Stubblefield & Mueller [46] studied t effects of variation in the NaC4. content of the medium on the growth, metabolism and chemical composition of HeLa cells in shortterm experiments (up to 13 days). Increases in the NaCl concentration from 120 mM to 220 mM resulted in a decreasedgrowt and at 250 or 280 mM NaCl, cell degen occurred [46]. Bobbins et al. 1371 hypertonic media depressthe ra sis of nucleic acids and protein HeLa cells; and induce a prop densation of the chromatin, described by others [l4, 15, 36, 461. Such Exptl Cell Res 70

318

D. 0. Schachtschabel & G. E. Foley

toxic and inhibitory effects of abnormal shifts in tonicity on the growth of mammalian cells have been known for some time [3, 12, 531; and Moscona et al. [29] stated “that tissues and cells which have to battle against unfavourable osmotic pressures sooner or later go down-hill and eventually succumb”. However, evidence will be presented herein that Ehrlich ascites tumor cells in culture can be adapted to long-term growth in hypertonic media. The present communication, the first in a series of reports dealing with the effects of different (hypertonic) concentrations of NaCl in the culture medium on the patterns of growth and metabolism of these tumor cells, describes their adaptation to growth in hypertonic media and the concomitant effects on morphology and growth rate. The capacity of cells to adapt to growth in such a grossly changed ionic environment might perhaps be regarded as a process of cellular ‘specialization’ similar in some respects to the process of “differentiation”. These studies were premised on two considerations. (a) Since the maintenance of physiologic concentrations of Naf and Kf on either side of the cell membrane is regulated by membrane-specific active transport processes (‘electrolyte pump’) (for review, cf [45]), cells adapted to growth in hypertonic media might be useful as in vitro model systems for the study of ion transport mechanisms and the effects of excess environmental salt concentrations on metabolism. Since studies with isolated tissues indicate a coupling between the active transport of cations and the transmembrane movement of sugars [5-8, 20-221, amino acids [l, 4, 8, 13, 16, 311,and possibly other substances, such as histamine, adrenalin, noradrenalin, and insulin [28, 30, 33, 561, it is possible that alterations in cellular metabolism (e.g., of carbohydrate and proExptl Cell Res 70

tein) could be induced by changes in the extracellular ionic environment. (b) Cell populations growing under controlled conditions in media containing increased salt content might be helpful in analysing the possible effects of changes in the ionic environment on genetic transcription and translation. There is increasing evidence that such environmental changes in the concentration of various inorganic ions may influence the DNA template and mitotic activity of mammalian cells [17, 18, 48-511, Furthermore, the pattern of nuclear RNA synthesis may be modified through exposure of isolated nuclei to different salts [24, 34, 35, 521, and it seems that the genomic activity of cells may actually be regulated by alterations in the electrolyte composition of their nuclei; and that the electrolyte balance in turn is subject to hormonal influences (for review, cf [ 191).Hence, specific in vivo effects of hormones might be mimicked by specific concentrations of electrolytes in vitro; such as, for example, the effects of ions on cellular differentiation in lower organisms [2, 541. In this connection, the studies of Siebert et al. [44] are of particular interest in that they suggest a direct communication for Naf ions between the extra-cellular space and the cell nucleus, suggesting that changes in the Na+ concentration in the cellular environment could exert direct effects on nuclear function. Thus, the ultimate purpose of these studies is the elucidation of regulatory mechanisms which govern the viability, growth and specific functions (‘differentiation’) of cells in culture. The studies have been presented in part as preliminary communications [38, 401. MATERIALS AND METHODS Media Eagle basal medium [lo] containing 0.1 mg streptomycin (Fa Farbwerke Hoechst) and 100 IU penicillin-G-sodium (Fa Griinenthal) per ml and supple-

Table 1. ‘Theoretical’ salt composition and salt content of media for ~~r~ic~ ascites tumor ceils cultivated in isotonic ‘control’ medium (ca 0.15 M) or hypertonic media with progressively increasing NaCl content Salt constituents of media (mmoles/l) Salt content of media @-Wl)

NaCl

NaHCO,

NaH2P04 x H,O

KC1

MgCl, x 6 H,O

CaCl,

0.15 ("control')

116

26.2

0.9

5.4

1.0

1.8

0.25 0.30 0.35 0.40 0.45

216 266 316 366 416 466

26.2 26.2 26.2 26.2 26.2 26.2

0.9 0.9 0.9 0.9 0.9 0.9

5.4 5.4 5.4 5.4 5.4 5.4

1.0 1.0 1.0 1.0 1.0 1.0

1.8 1.8 1.8 1.8 1.8

CL50

1.8

‘Control’ medium was Eagle basal medium [lo] supplemented with 10 % undialysed fetal calf serum. Media with increased NaCl content (0.25, 0.30, 0.35, 0.40, 0.45, 0.50 M) were prepared by addition of appronriate amounts of NaCl to ‘control’ medium. Composition is termed ‘theoretical’, since figures for ‘control’ media are those of Eagle basal medium [lo]. Concentrations were not corrected for the slight changes caused by addition of undialysed fetal calf serum. mented with 10 % fetal calf serum IEBM] was obtained from GIBCo (Grand Island, N.Y.). A stock solution of ca 2.15 M NaCl (Suprapur; Merck, Darmstadt; BRD; MW = 58, 44) was prepared by addition of 50 ml of EBM to 6.08 g of autoclaved NaCl (final volume ca 52 ml). Since EBM contains Earle salts (table 1) and 10% undialysed fetal calf serum, the actual NaCl concentration of the stock solution mav be slightly less than 2.15 M, since the molarity of ali salts-in the culture medium is 0.15 M (table l), and the total salt concentration in the serum was presumed to be 0.15 M. Media with increased NaCl content (table 1) were prepared by appropriate dilution of this NaCl stock solution with EBM, e.g., 14.3 ml of stock NaCl solution added to 100 ml of EBM results in a final salt concentration of 0.40 M. The salt concentrations (in M) and the ‘theoretical’ salt composition of these media are listed in table 1.

equal volumes of a pooled cell suspension in 2-ounce pharmacy bottles. -Medium changes (with IO ml volumes of medium) were made at 37°C at frequencies indicated in the individual experiments. Following incubation, replicate cultures were decanted, the monolayer of cells washed twice for 2 min at room temperature with Earle salt solution [ll] adjusted with NaCl to equal the salt content of the medium in which the cells were grown, and the total protein content of the airdried monolayer determined-by the Lowry method 1271as modified by Oyama & Eagle [32], using bovine serum albumin-&ma, St Lo& MO.) as a standard. Photomicrographs of monolayer cultures growing in T-15 culture flasks (Belico, Vineland, N.J.) were made in an inverted microscope (Standard UPL; Carl Zeiss Inc.) with an attached camera (E. Leitz, Inc.). The ‘stereo-like appearance’ of the pictures was obtained by turning the condenser slightly ant of focus to obtain oblique illumination..

Cell cultures The origins, isolation and maintenance of these Ehrlich ascites tumor cells have been described elsewhere [39, 411. Approx. 90 % of the cells are hyperdinloid (42-45 chromosomes),,, with typical marker __ cb~omosomes 1413. Monolaver cultures for the present exueriments were prepared by seeding l-5 k lo5 cells-in 5 ml volumes of EBM in 2- or 4-ounce pharmacy bottles (Fa Federated Distributors) in which the atmosphere was replaced with a mixture of 7 %COz, 20% 0% and 73 % N,. Subcultures were made by scraping the cells from the glass with a ‘rubber policeman’. Growth curves were obtained by determination of the total cellular protein content of cultures incubated for various lengths of time at 37°C. In these experiments, cultures were seeded by distribution of

Adaptation of cells to growth in media with increased NaCl content A schematic outline of several method:? by which Ehrlich ascites tumor cells were adapted to survival and multiplication in hypertonic media is indicated in fig. 1. The salt compositions of these media are listed in tab1.e 1, and the salt concentrations (in Ma/n> are indicated in the squares in fig. 1.

320 D. 0. Schachtschabel & G. E. Foley

I

6

Fig. 1. Schematic outline of sequential propagation of Ehrlich ascites tumor cells in media containing increased NaCl concentration (cf table 1). Numbers at arrows denote number of days between increases in salt concentration in medium, resp. transferring cells. Medium changed at 48 h intervals. Numbers in squares indicate salt concentration (in M) in medium. A, B, and C depict three different methods of ‘adaptation’ to growth in hypertonic media. In each case (from left to right), subcultures were grown 7 days in isotonic control medium (0.15 M), reaching ‘stationary’ growth phase before salt concentration was increased to 0.25 M; 2 days later to 0.30 M; 2 days later to 0.35 M, etc.

The most effective, and hence most frequently used method of adaptation is depicted in fig. 1B. Subcultures were grown for 1 week (with medium changes every second day) in isotonic control medium (0.15 M medium), reaching a nearly stationary population of ca 20 x lo6 cells/Counce pharmacy bottle. The salt concentration of the culture medium was then increased stepwise to 0.40 M by means of 1.5ml medium changesevery 2 days, such increases resulting in the loss of ca 80% of the cells in the cultures. In the following 8 days, the salt concentration in the cultures was decreasedstepwise to 0.25 M by medium changes every second day, and during the next 8 days, the remaining cells (ca 2-5 x 106) which had then ‘recovered’ increased to ca 15-20 X lo6 cells per bottle. The salt conExptl Cell Res 70

centration was again increased successively to 0.30 M and to 0.35 M in 10 days with 15 ml medium changes every 2 days, and the cells subcultured in 0.35 M medium. After 2 weeks, with medium changes every other day, these subcultures contained confluent monolayers of cells which could be readily subcultured. Following 5 serial subcultures within 3 months, the salt concentration was increased to 0.40 M, and after 5 additional subcultures within 7 months, the salt concentration was increased to 0.45 M, and subsequently to 0.50 M (fig. 1); the maximum tolerable salt concentration attained thus far. In other experiments (fig. 1A) attempts to adapt cells to grow in the 0.35 M medium without intermediate reduction of the salt concentration resulted in the loss of most

Cultivation of tumor cells in hypertonic media

32i

Fig. 2. (a-d) Monolayer cultures of Ehrlich sscites tumor cells growing in media with different XaCI contents. x 150. (a) ‘Control’: c&s in Eagle basal medium supplemented with 10 % fetal calf serum (salt concentration ca 0.15 M). (b-d) Cells in same basal medium, except for increased NaQ content: 0.35 M (6); 0.40 M (c) and 0.45 M (d). (e) Cells ‘re-adapted’ to growth in control. medium after serial cultivation in medium with increased NaCl content (0.40 M). Same culture as that in fig. 2c (I4 days later): the salt concentration of the medium was lowered within 8 days from 0.40 to 0.15 M. Photograph was taken 6 days after the cells bad been ‘readapted’ to growth in 0.15 M medium.

(ca 99 X) of the cell population. However, after ca 2 months, several colonies of cells developed, and after 3 months these cultures contained a confluent monolayer which could be subcultured. Procedures B and C (fig. 1), with intervening reduction of the salt concentration to permit ‘recovery’ of the surviving cells are, however, more reliable methods for the derivation of cells which can survive and grow in hypertonic media.

Designation of ~igh-§a~t-t~~e~~~t ceils Cultures serially propagated in media with the salt concentrations of 0.15 M (“control’, .40, 0.45 isotonic medium), 15, EDand 0.50 M, were 0.25, ED-0.30, etc. AU the intermediate cultures of these high-salt-tolerant cel!s been maintained; thus far, for example, ED0.35 cells have been maintained in culture ca 3 years and have been subcultured more

322

D. 0. Schachtschabel h G. E. Foley

30

3. Representative growth-curves of log-phase cultures of Ehrlich ascites tumor cells in media containing different concentrations of NaCl. Abscissa: time of incubation (hours): ordinate: ma oroteini culture. T, doubling‘time.“O-0, ED -0.i5 (T= 16h); x--,ED-0.25(T=21 h); A-A,,D-0.30 (T=23 h); O-O, ED-O.35 (T=38h); V-Y, ED -0.40(2-=53h); n -w,ED-O.~~(T=~~~).ED0.15 cells (‘control’) in Eagle basal medium supplemented with 10 % fetal calf serum (salt concentration ca 0.15 M). ED-0.25, -0.30 up to -0.45: highsalt-tolerant cells in media with increased NaCl content (salt concentration: 0.25; 0.30 UD to 0.45 M). Each point represents the mean of at least 2 replicate cultures (SD. less than ilO%). Fig.

respective media. When the monolayer is scraped into the medium, the ED-O.15 cells are suspended predominantly as single cells, while the high-salt-tolerant cells often stick together and form aggregates. These morphological alterations are regarded as adaptive changes, since ‘re-adaptation’ of such high-salt-tolerant cells to growth in isotonic media results in the re-appearance of an ‘epithelial-like’ morphology (compare fig. 2e with 2~). The cells in fig. 2e (designated ED-N-O. 15) were derived from a culture of ED-O.40 cells (fig. 2~) which had been Ye-adapted’ to growth in isotonic media, and it is evident that their morphology now resembles that of the original ED-O.15 cells (compare fig. 2e with fig. 2a). Growth

Representative growth curves of exponentially growing cultures are illustrated in fig. 3. After seeding the cultures at 37°C 12-24 h for ED-O.15 and ED-O.25 cells, ca 36 h for ED-O.30 cells, ca 48 h for ED-O.35 cells, ca 72 h for ED-O.40 cells, and ca 96 h for ED0.45 cells had to pass before the respective than 60 times, and ED-O.40 and ED-O.45 cultures entered the logarithmic growthcultures have been subcultures ca 20 times phase. Therefore, in these experiments (fig. 3) ED-0.15, ED-0.25, ED-0.30, and ED-O.35 over a period of more than 2 years. cells were seeded 48 h before O-time, and Morphology ED-O.45 cells 96 h before O-time (fig. 3). The Ehrlich ascitestumor cells growing in isotonic first medium changes (10 ml) was made at control medium (ED-O.15 cells) exhibit an O-time (fig. 3) and repeated at 24 or 48 h ‘epithelial-like’ morphology (fig. 2a) and are intervals depending on the degree of acidifinot ‘contact inhibited’, as evidenced by the cation of the medium. These growth curves (fig. 3) clearly demon‘piling up’ of cells in the monolayer (fig. 2a). In contrast, cells growing in media with an strate the increasing population doubling increased NaCl content are larger, flattened time consequent to increasing salt concentraand extended, exhibiting in general a more tion of the medium (table 2). Under these ‘fibroblast-like’ appearance (fig. 2 b-d). This experimental conditions, these doubling times ‘fibroblast-like’ morphology is more pro- appeared to be independent of differences in nounced in the case of ED-0.35, ED-0.40, the duration of the log-phase and time in ED-0.45, and ED-O.50 cells, and persists in culture; for example, the average doubling subcultures of these various cell lines in their time of ED-O.35 cultures was ca 36 h after Exptl

Cell Res 70

Cultivation

6 months, 1,2, and 3 years in 0.35 M medium. However, Ye-adaptation’ of high-salt-tolerant cells (e.g., ED-O.40 cells) to growth in isotonic medium resulted in an acceleration of growth, the average population doubling time of such cultures approaching that of the original parent culture-14 h as compared with 15 h (table 2). DISCUSSION §ince the inhibitory and toxic effects of increased salt concentrations result from interference with DNA synthesis and mitotic processes [9, 14, 15, 36, 37, 46, 531, in the gresent studies increases in substrate NaCl concentration were initiated in cultures presumed to be in stationary growth-phase; stepwise increases to high salt concentrations being followed by a gradual decrease in concentration to select and enrich high-salttolerant cells, and to relieve the surviving cells from the possible ‘toxic’ effects [3, 9, 12, 14, 15, 36, 37, 46, 531 of high salt concentrations. The gradually increasing population doubling time concomitant with increasing substrate salt concentration suggests that changes in the extra-cellular ionic concentration may reflect cellular metabolic alterations (e.g., cell membrane changes) which influence the growth rate. It should be noted, however, that these alterations in cell morphology and growth pattern appear to be adaptive changes induced by increasing salt concentrations, since reduction of the NaCl concentration to the original level in Eagle basal medium resulted in a reversion of morphology and growth rate to that of Ehrlich ascites tumor cells maintained in unaltered Eagle basal medium. Cells derived from well-defined parent cultures and adapted to grow in media altered with respect to a single essential salt component such as NaCl should provide an in vitro model system for studying the effects

of

tumor

cells

in

hypertonic

media

323

Table 2. Average population doubling times in log-phasecultures of Ehrlich ascites tumor cells in media containing different ~o~~e~t~~t~o~~ of N&k Cell CUlturea

Referred to douSing Doubling time time of ED-O.l5 (hours) culture as 1.0

ED-O.15 ED-O.25 ED-O.30 ED-O.35 ED-O.40 ED-O.45

15 (I2-18)b 20 (17-23) 23 (21-25) 36 (33-39) 53 (X-60) 63 (55-70)

ED-N-0.15C

14(12-16)

1.QO 1.33 1.53 2.40 3.53 4.20 0.93

a Cultures identified in fig. 3. b Range in hours. ’ Cells ‘readapted’ to growth in isotonic control medium after serial cultivation in medium with increased MC1 content (0.40 M).

of such a substrate alteration on cel~~~a,r metabolism and the biological an mica1 mechanisms underlying cellular transport, uptake and metabolism of this component. Further, if cultivation in SW strate results in permanent biolo biochemical differences between genetically ‘identical’ cell lines which differ only with respect to tolerance to increased eoncentrations of NaCl, such cellular alterations may be directly related to the altered ionic composition of the medium. Contrary to the expectation that exposure to hypertonic media might result in shrinkage, it is of interest that these h salt-tolerant cells were characterized by an increase in cell surface, and actually have an increased protein content [42]. These highsalt-tolerant cells also seem to stick more tightly together than the parent cells; and whether this increased ‘adherence’ is a reflection of possible ‘i~ter6on~e~tio~s’ of the cells in the monolayer (e.g., these cells coul be ‘embedded’ in a matrix of mucous material synthesized, secreted, and layered around

324

D. 0. Schachtschabel & G. E. Foley

the cell surface as ‘protection’ against the high-salt content of the substrate) remains to be determined. The authors thank Mrs E. Krutzsch and Miss J. Ferro for excellent technical assistance. These studies were supported in part by research grants from Deutsche Forschungsgemeinschaft to D. 0. Schachtschabel, and research grants C-6516 from the National Cancer Institute, and FR-05526 from the Division of Research Facilities and Resources, National Institutes of Health to The Children’s Cancer Research Foundation. G. E. F. holds Research Career Award-K6-CA-22, 150 from the National Cancer Institute, National Institutes of Health.

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