induction of anchorage-independent growth in human...

[CANCER RESEARCH 41. 1620-1627. May 1981]0008-5472/81 /0041-0000$02.00

Induction of Anchorage-independent Growth in Human Fibroblasts byPropane Sultone1

K. Charles Silinskas, Suzanne A. Kateley, John E. Tower, Veronica M. MÃ¤her,and J. Justin McCormick2

Carcinogenesis Laboratoryâ€”Fee Hall, Departments of Microbiology ano Biochemistry, Michigan State University, East Lansing, Michigan 48824

ABSTRACT

We have demonstrated a dose-dependent increase in thefrequency of diploid human cells capable of anchorage-inde

pendent (Al) growth after treatment with the carcinogen propane sultone, followed by exponential growth to allow fullexpression of this phenotype (8 to 13 population doublings).Exposure to these same concentrations of propane sultonealso resulted in a dose-dependent increase in the frequency of6-thioguanine-resistant cells in the population. Procedures

such as synchronization of cells and treatment just after theonset of DMA synthesis or the use of special selective mediumwere not essential for this induction. A very low frequency ofcells with the Al phenotype was found in the control population(background). Cells which exhibited the Al phenotype spontaneously or after carcinogen treatment retained the characteristic over as many generations as tested (>13). The datasuggest that Al growth is the result of a mutational event.

INTRODUCTION

A number of workers have demonstrated the existence of ahigh correlation between the ability of animal cells transformedin culture by viruses or chemical carcinogens to form coloniesin semisolid medium (Al3 growth) and their ability to form tumors

in appropriate host animals (1, 3, 5, 6, 12, 16, 17, 23). It hasbeen shown that normal diploid human cells can be induced toform colonies in semisolid medium by treatment with SV40 (seefor example Refs. 8 and 25) and more recently by exposure tochemical carcinogens (7, 15, 20) or UV radiation (26). However, with the exception of Milo and DiPaolo (20), these investigators failed to obtain tumors following injection of the progeny of these human cells into athymic mice (12).

Because of the importance of understanding the process ofmalignant transformation of diploid human cells and the significance of a possible connection in human cells between Algrowth and tumorigenicity, we have reexamined the role of the

' Supported in part by Department of Health and Human Services Grant CA

21289 from the National Cancer Institute and by a grant from the Elsa U. PardeeFoundation.

2 To whom requests for reprints should be addressed.3 The abbreviations used are: Al, anchorage independent; sMEM. Eagle's

minimum essential medium supplemented with 25 mM 4-(2-hydroxyethyl)-1-pi-perazineethanesulfonic acid (pH 7.4), 10% fetal calf serum (K. C. Biological, Inc..Lenexa, Kans.). 1 mM sodium pyruvate, 0.2% sodium bicarbonate, 1x nones-sential amino acids (Flow Laboratories. Inc., McLean, Va.), and 50 pg of genta-micin sulfate (Sigma Chemical Co., St. Louis, Mo.) per ml; MEM, Eagle's minimumessential medium; FCS, fetal calf serum; MNNG, N-methyl-N'-nitro-N-nitrosogua-

nidine; PBS. phosphate-buffered saline [containing 0.8% NaCI, 0.02% KCI,0.12% Na2HPO4, 0.02% KH2PO4 (pH 7.2)); 9sMEM, Eagle's minimum essential

medium supplemented with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesul-fonic acid (pH 7.4), 20% fetal calf serum. 1 mM sodium pyruvate, 0.2% sodiumbicarbonate, 9 x nonessential amino acids, 2 x vitamins (Flow Laboratories, Inc.).and 50 ^g of gentamicin sulfate (Sigma) per ml; TG, 6-thioguanine.

Received September 15, 1980; accepted January 22, 1981.

various steps prescribed by Milo and DiPaolo (20) for theinduction of anchorage independence by chemical carcinogens. According to their report, induction of Al growth is theresult of a complex series of steps. Fibroblasts must be of earlypassage. They must be synchronized, pretreated with suchagents as insulin or anthralin, exposed to the mutagen/carcin-

ogen for 12 hr in the middle of DMA synthesis, and thensubcultured for ~21 population doublings in medium contain

ing increased concentrations of nonessential amino acids andvitamins before being assayed for Al growth in low-calcium

medium. They reported frequencies ranging from 10 to 100 Alcolonies/106 cells plated into soft agar in the first cycle.

Nodules were sometimes obtained from cells isolated from asingle cycle in agar and propagated to large populations, butmuch higher frequencies of tumors resulted from cells recycled2 more times into soft agar. In contrast, Freedman and Shin (7)induced Al growth in human fibroblasts by treating them witha mutagen and assaying them 7 days later. These latter workersreported obtaining frequencies of 3 to 10 Al colonies/105 cells

plated. However, they failed to obtain tumors upon injection ofthe progeny of these cells into athymic mice. To investigate thereasons for these different findings, we attempted (a) to repeat,in principle, the protocol of Milo and DiPaolo (20); (o) toinvestigate which steps of their protocol were essential forcarcinogen induction of Al growth and which were either unnecessary or had only a slightly enhancing effect; and (c) todetermine the kinetics of expression of the Al phenotype inhuman cells.

MATERIALS AND METHODS

Cell Cultures and Medium. Foreskin tissue from newborninfants was minced, and the cells were dispersed with colla-

genase (Sigma Chemical Co., St. Louis, Mo.) (22). The cellswere pooled and used for the experiments described. Unlessotherwise indicated, cells were cultured in sMEM in a humidified incubator containing 5% CO2:95% air at 37Â°. A human

kidney carcinoma cell line (Hs835T) obtained from the NavalBiosciences Laboratory, Naval Supply Center, Oakland, Calif.,was used as a positive control in the assays for Al growth andtumor formation.

Treatment of Cells after the Onset of DNA Synthesis. Cellswere synchronized by the method of Milo and DiPaolo (20).Briefly, the medium on exponentially growing foreskin-derived

cells was changed to MEM lacking arginine and glutamine andsupplemented with 10% dialyzed FCS. After 24 hr, the mediumwas changed to sMEM containing 1 /ig of anthralin per ml(Aldrich Chemical Co., Inc., Milwaukee, Wis.). Ten hr later, anappropriate dose of carcinogen dissolved in acetone (propane sultone) or water (MNNG) was added to the 20 ml ofmedium in the flask. The final concentration of acetone in themedium was 0.5% or less. After 14 hr, the medium was

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Al Growth of Human Fibroblasts

removed, and the cells were rinsed with PBS, trypsinized(0.25%), and subcultured at a 1:2 dilution into flasks containingsMEM or 9sMEM,Just before they reached confluence, thecells were trypsinized and subcultured in the same medium tomaintain exponential growth.

Assay for Cytotoxicity. At the end of the 14-hr exposure tocarcinogen, when cells were trypsinized for subculturing 1:2,an aliquot of the cell suspension was diluted appropriately, andthe cells were plated in 60-mm dishes at cloning densities to

determine the cytotoxic effect of the treatment from the loss ofcolony-forming ability (18). The cloning efficiency of the treated

population divided by that of the untreated synchronized population was determined and is expressed as a percentage.

Agar Preparation. Bacto-agar (Difco Laboratories, Detroit,Mich.) was prepared for use by washing 15 times with distilledwater. Agar (300 g) was added to 15 liters of glass-distilled

water and stirred for 6 hr. The agar was allowed to settleovernight, and the water was removed by aspiration. Freshdistilled water was added, the washing was continued as aboveexcept that the stirring was reduced to 2 hr, and 4 or more hrwere allowed for settling before aspiration of the water. Afterthe 15th wash, the water was removed from the agar suspension by filtering through Whatman No. 1 filter paper, and theagar was spread on a clean surface and allowed to dry. Theagar was then ground into a fine powder, dried completely, andstored until use.

Assay of Anchorage Independence. The frequency of cellscapable of Al growth was assayed using conditions similar tothose of MacPherson (17). Briefly, bottom agar medium wasprepared by combining one volume of washed agar (1 % indistilled HjO) with 1 volume of double-strength MEM containing

20% PCS (v/v) and 20% tryptose phosphate broth (v/v) anddistributing 5 ml of this mixture into 60-mm-diameter dishes.For top agar medium, one volume of cells in sMEM at 37Â°wascombined with 2 volumes of bottom agar medium at 40Â°.Three-mi aliquots of this top agar (containing 103 to 105 cells)

was gently overlaid onto the solidified bottom agar in thedishes. The final concentration of agar in the top layer was0.33%. For the experiments described here, 1 ml of sMEM wasgently added to the cultures 24 hr after plating the cells. After2 weeks, an additional 1 ml of sMEM was added to make upfor evaporation. (Our recent studies indicate that refeedingabout once a week after plating is more satisfactory.) Cellswere incubated at 37Â°with a humidified atmosphere of 97%

air:3% CO2. After 3 weeks, the number of macroscopic colonies composed of 1000 or more cells (>0.2-mm diameter) were

counted using a dissection microscope. Recovery of cellsexhibiting Al growth was accomplished by gently removing alarge number of the colonies from dishes containing soft agarwith a Pasteur pipet and plating them into a flask containing 5ml sMEM. After 2 to 3 days, the multicellular colonies attachedto the plastic, and the cells began to grow out. These cellswere grown to suitably sized populations (>107 cells in total)

and then reassayed for Al growth and/or injected into athymicmice to test for tumorigenicity.

Flow Cytometry. Cells (~1 x 106) were prepared for flow

cytotometry (9) by removing the medium and washing the cellswith PBS followed by addition of 0.25% trypsin. When the cellsshowed surface alteration, but before they detached, the trypsin was removed, 5 ml of PBS were added, and the solutionwas gently pipeted until a single-cell suspension formed. Then,

95% ethanol was added to make a final concentration of 70%ethanol, and the cells were stored in this solution at 4Â°for not

more than 4 weeks. When flow cytometry analysis was to becarried out, the cells were centrifuged at 500 x g for 10 min,and the supernatant was discarded. The pellet was suspendedin 5 ml of a solution of 0.1 M Tris-HCI (pH 7.5):0.1 M NaCI:0.003 M EDTA containing 2.5 mg of 4'-6-diamidino-2-phenylin-

dole (Sigma) per 1000 ml. An Ortho ICP-22 flow cytometer was

used for the analysis of DMA content/cell. To remove anyclumps, the cells were filtered through a Korb filter (OrthoLaboratories, Boston, Mass.) prior to flow cytometer analysis.

Mutagenicity Assay. The assay for the induction of -TG-

resistant cells was carried out as described previously (18).Essentially, cells were kept in exponential growth for 6 to 8days (i.e., 5 to 6 population doublings) following mutagentreatment, and then 1 to 2 x 106 were selected at a density of500 cells/sq cm in Ham's F-10 medium lacking hypoxanthine

and supplemented with 40 fiM TG and 10% FCS. The cellswere incubated at 37Â°with a humidified atmosphere of 95%

air:5% CO? and were refed once with selection medium after10 days. The dishes were stained with mÃ©thylÃ¨neblue at 18 to21 days. Macroscopic colonies composed of >100 cells werecounted, and the frequency was determined directly from thenumber of colonies per number of clonable cells plated as wellas indirectly by the P(O) method4 as described (18). The

cloning efficiencies of the cells at selection averaged -40%.

Tumor Assay. To examine the ability of cells to form tumors,1 to 10 x 106 cells in 0.2 ml of sMEM were injected s.c. into

the flank of male BALB/c (nu/nu) athymic mice (Sprague-

Dawley, Madison, Wis.) 5 to 10 weeks of age. Similar numbersof Hs835T or untreated human cells (either derived from agarcolonies taken from controls or, more commonly, cells fromuntreated cultures) were also injected as the positive andnegative control, respectively. All mice were reared in hoodswith Hepa filtered air and were completely isolated from otheranimals. Mice were examined weekly for s.c. tumors.

RESULTS

Preliminary Study with MNNG. Before attempting to maximize the efficiency of induction of anchorage independence indiploid human fibroblasts by chemical carcinogens, we undertook a preliminary study patterned as closely as possible uponthe protocol developed by Milo and DiPaolo (20). This studywas greatly facilitated by Professor George Milo, The OhioState University, who provided detailed advice on various partsof the procedure and was available for consultation. For thisinitial attempt to reproduce, in principle, the results of Milo andDiPaolo (20), we used MNNG as the mutagen/carcinogensince we5 and others (11) had found it to be a strong mutagen

in diploid human fibroblasts, and it had been used successfullyby Milo and DiPaolo (20) to induce Al growth.

Early-passage cultures of fibroblasts (passage 3) were synchronized as described in "Materials and Methods," treated

with anthralin (1 /jg/ml), and exposed to MNNG (6.5 and 9.3/iM) beginning 10 hr after release of the cells from the G,-Sblock. Fourteen hr later, when the cells were trypsinized anddiluted 1:2 into the selective medium 9sMEM as prescribed

' P(O) method, determining the chance of a mutant per dish from the number

of dishes with no mutants, assuming a random distribution.5 V. M. MÃ¤her,and J. J. McCormick, unpublished results.

MAY 1981 1621



K. C. Silinskas et al.

(20), we assayed a portion for the percentage of survival ofcolony-forming ability. Although not part of the procedures of

Milo and DiPaolo (20), this assay which we use routinely in ourresearch as an accurate method of comparing the actual biological doses received by cells in one experiment with that ofanother (18) did not interfere with following their protocol. Theresults indicated survivals of -14 and -4%, respectively. Although Milo6 indicated that in his experience survival levels

greater than 50% were optimal, we continued with the experiment because consultation with him revealed that in his laboratory cell survival was estimated by dye exclusion techniquescarried out as described in Ref. 21 on populations only after amuch greater time period had elapsed following the initialexposure to the cytotoxic agent. Therefore, direct comparisonsof his cell survival and ours were not possible.

The surviving cells were subcultured 6 times using 1:10dilutions at each passage as prescribed (20). Contrary to whatMilo6 suggested would occur, the untreated population flour

ished in 9sMEM and gave no evidence of being at a selectivedisadvantage. After ~21 population doublings (i.e., six 1:10

passages), treated and control populations were assayed forAl growth (Agar 1) using the procedure described in "Materialsand Methods." It must be noted here that for the assay of cells

capable of growth of soft agar we used a method modifiedslightly from that of MacPherson (16) but differing significantlyfrom that of Milo and DiPaolo (20). This was because we foundthrough reconstruction studies using human carcinoma-derived cells (Hs835T) that the number of Al colonies formed inagar medium prepared following Milo's formula was drastically

(i.e., 50-fold) lower, and we wanted to enhance our chances of

observing Al colonies as much as possible in this pilot experiment. The experimental cells formed colonies in agar with highfrequency (1.8 and 3 x 10~3, respectively). The untreated

cells did so only at a much lower frequency. Large-sized

colonies were isolated from the soft agar, propagated into largenumbers of cells, and assayed a second time (Agar 2), and theprocess was repeated a third time (Agar 3) as prescribed (20).The frequency of Al colonies observed in Agar 2 and 3 for bothtreated and untreated cells ranged from 1 to 4 x 10~2.

Cells isolated and propagated from Agar 1,2, and 3 werealso assayed for tumorigenicity by s.c. injection into athymicmice (1 to 6 x 106 cells/mouse; 6 mice/assay). Hs835T cells

derived from a human tumor were similarly injected as positivecontrol. The latter cells always produced nodules ranging from0.2 to 1 cm in diameter within 3 to 4 weeks after injection.None of the control cells gave rise to nodules. A number ofanimals given injections of experimental cells, i.e., progeny ofMNNG-treated cells isolated from soft agar and propagated to

large populations, developed palpable nodules approximately0.6 cm in diameter. These nodules were not excised but wereallowed time to enlarge. Instead, they regressed over a periodof â€”10days. However, one mouse injected with progeny of

cells exposed to the lower dose of MNNG which had beenderived from Agar 2 developed a nonregressing tumor ~40

days following injection. After 1 month, this nodule was excisedand characterized independently by several pathologists as aclassic fibrosarcoma. This tumor was composed of cells with ahuman karyotype. It should be noted that the athymic micewere not X-irradiated as prescribed (20) because, when we did

6 G. Milo, personal communication.

irradiate according to their protocol, 50% of the animals died.Moreover, Kakunaga (13, 14) had obtained tumors with progeny of carcinogen-treated human cells in unirradiated mice,

and more recently Borek (4) has also done so.Studies Designed to Analyze the Steps Required for In

duction of Anchorage Independence. The basic transformation protocol of Milo and DiPaolo (20) can be considered asconsisting of 2 parts, namely, (a) induction of ability to grow insemisolid media and (b) induction of ability to form tumors.Their data (20) suggest that these are 2 independent processesor steps but that selection of Al cells by semisolid mediumenriches the population for cells which have been or will bechanged into tumor-forming cells. Therefore, once we had

succeeded in the induction of anchorage independence byfollowing, in principle, their procedures (20), we undertook toexamine the steps involved (a) to determine which were essential for the process and (b) to see if we could improve theefficiency of the process itself. After consultation with Milo,6

propane sultone was chosen as the carcinogen/mutagen because it had proved most active in his experiments (20). Thefirst question asked was is it necessary to synchronize the cellsand expose them to the agent during DNA synthesis or wouldtreatment of populations in exponential growth be as efficient?The second question asked was whether growth of cells in9sMEM was required for induction of Al growth or at leastafforded Al cells some selective advantage during the periodfollowing carcinogen treatment. A third question asked waswhether it was necessary to allow ~21 population doublings

(six 1:10 passages following the initial 1:2 dilution) in order toobserve high frequencies of Al cells.

Early-passage cells (passage 3) were exposed to 3 concen

trations of propane sultone, those which we determined fromcytotoxicity assays would reduce the cell survival to ~80, 40,

and 10%. One set of cells was not synchronized but exposedto various concentrations of propane sultone for 14 hr inexponential growth. The other 2 sets were synchronized,treated with anthralin, and exposed to the various concentrations of propane sultone. However, at the time of the firstdilution of 1:2, one set of these synchronized cells was propagated in sMEM and the other in 9sMEM. The control seriesand the 3 experimental series, each composed of 3 separatepopulations, were assayed for Al growth at the end of three 1:10 passages as well as after the prescribed six 1:10 passages(20). The results are shown in Chart 1 and Table 1, Columns6 and 9. The frequencies of Al cells induced by propanesultone were concentration dependent. Furthermore, the frequencies observed in the experimental populations assayedafter -11 population doublings were significantly (10-fold)higher than those seen after ~21 doublings. The data indicated

that the enhancing effect resulting from synchronization of thepopulation so as to expose cells during S phase was about25%. Similarly, propagation of the mutagen-treated cells in9sMEM gave ~25% enhancement over sMEM (Table 1).

Stable Inheritance of the Al Phenotype. To test whethercells which had acquired the property of Al growth maintainedit as a stable characteristic, we isolated cells growing ascolonies in Agar 1, propagated them to large populations asmonolayers in flasks, and retested them for their ability toproduce colonies in soft agar (Agar 2). Cells that grew ascolonies in Agar 2 were also reisolated, grown up in largepopulations in plastic flasks, and retested for growth in agar





Table 1Frequency of Al colonies per 10s cete plated in sort agar

Concentration of

propaneâ€¢ul-tone(fig/ml)0102535Cloningeffi

ciency0.110.240.0930.210.0340.0750.0100.015Sur

vivalas%ofcon

trol1001008686313196Growth

condition at timeoftreatmentSynchronousSynchronousRandomSynchronousSynchronousRandomSynchronousSynchronousRandomSynchronousSynchronousRandomCulture

mediumfromtreatmentup

toAgar1sMEM9sMEM9SMEMsMEM9sMEM9sMEMsMEM9sMEM9sMEMsMEM9sMEM9sMEMAssayed

inAgar1after1

1 doublings"9

Â±3e14

Â±36Â±1520

Â±40810Â±40520Â±501

,300 Â±401.700Â±801

,400 Â±3002,400

Â±702,600Â±1202.000Â±180Isolated

fromAgar1, propa

gated,0 andreas-sayedin Agar2Â°ND'6,700

Â±1,200NOND9,200

Â±900NDND9,500

Â±1,000NDND9,000

Â±1,000NDIsolated

fromAgar2, propa

gated," andreas-sayedin Agar3CND8,500

Â±1,400NDND1

9.000 Â±1,200NDND20,000

Â±1.400NDND20,000

Â±1,600NDAssayed

inAgar1after

21doublings"5

Â±119Â±14Â±11

30 Â±10150 Â±2090

Â±10190

Â±20200Â±30160Â±20270

Â±30320Â±20230Â±20Isolated

fromAgar1. propa

gated,andreassayedinAgar

2CND7,300

Â±900NDND9,400

Â±900NDND9,300

Â±1,000NDND9,600

Â±1,000NDIsolated

fromAgar2,propa-gated,11

and reassayed in Agar3CND9,700

Â±1,300NDND20.000

Â±700NDND19.000

Â±1.000NDND19.000

Â±1.500ND

10s cells assayed/dish; 10 dishes/determination.6 The cells which grew out of the isolated colonies were propagated in sMEM for >10 population doublings.c 10" cells assayed/dish; 20 dishes/determination.d Cells were synchronized, and the carcinogen was added shortly after the onset of S phase.e Mean frequency of Al colonies per 105 cells Â±S.D.' ND, not determined.

â€ž 3000

â€” Râ€”-^--^^^1- Doublings1 1 Till

IO 20 30 40PROPANE SULTONE CONCENTRATION (u.g/ml)

Chart 1. Frequency of Al colonies of human cells assayed after 11 or 21population doublings following treatment with propanesultone. Cells were synchronized (20) and treated with carcinogen shortly after the cells had begun DMAsynthesis (synchronized) or treated as exponentially growing cultures (random).Following a 14-hr exposure to carcinogen, both populations were trypsinized,diluted 1:2, and replated into flasks in 9sMEM medium. From then on, bothpopulations were kept in exponential growth and were assayed for anchorageindependence (105 cells/dish; 10 dishes/assay) after 3 or six 1:10 dilutions.

(The number of population doublings is only an approximation.) The backgroundfrequency of Al cells in these control populations, which were handled exactly asthe treated cells with the omission of carcinogen, averaged 11 x 10 5.

(Agar 3). The frequency of Al colonies produced in each assayis shown in Table 1. The data listed in Column 6 (Agar 1) aretaken from progeny allowed to undergo an initial expressionperiod of â€”11 population doublings; those listed in Column 9(Agar 1) are from progeny which were allowed ~21 doublings.The results with Al cells recycled into Agar 2 clearly indicatedthat cells exhibiting Al growth maintained this as a stablecharacteristic since such cells showed a greatly enhancedfrequency of Al colonies. Note that for cells assayed in Agar 2there is no significant difference between the various frequencies, neither those of the experimental cells nor the controlcells. This result is to be expected if cells which attain anchor

age independence spontaneously or as the result of exposureto a mutagen inhert this phenotype as a stable characteristic.The frequencies of Al cells in the experimental populationsassayed in Agar 3 were ~2-fold higher than in the correspond

ing Agar 2 assays. That a similar increase was not found withthe progeny of the untreated population probably reflects differences in the number of population doublings allowed between assays since no special attempt was made to keep thisnumber uniform. Each set of cells was assayed in Agar 3whenever the size of the population was judged sufficient forour purposes.

Assay of the Optimal Time of Expression of Al Growth. Inthe light of these results which demonstrated a 10-fold higher

frequency of cells capable of Al growth in progeny assay after-11 population doublings than after 21 doublings, it was ofinterest to determine the time of optimal expression of thisphenotype. Chart 2 shows the kinetics of expression of Algrowth by progeny of cells treated at passage 10 with propane sultone. These cells were synchronized, treated withanthralin, and exposed as described in "Materials and Methods" to propane sultone at a concentration of 21.5 jug/ml.

After treatment, however, the cells were plated in sMEM insteadof in 9sMEM. The results showed that 8 to 11 populationdoublings were required for the maximum expression of Algrowth and that, after remaining approximately the samethrough 13 doublings, the frequency of cells capable of Algrowth declined rapidly. The latter result would be expected ifAl cells were at a selective disadvantage.

Comparing Induction of Anchorage Independence and TGResistance. We also carried out mutagenicity studies withpropane sultone to determine whether the concentrations required for the induction of TG-resistant mutants were similar to

those required for the induction of Al growth in these cells. Theresults, shown in Chart 3, demonstrated that this was indeedthe case. Cells from passage 11 were synchronized as described, exposed as usual to propane sultone at the indicated

MAY 1981 1623




100

PROPANE SULTONECONCENTRATION (u.g/ml)

.0 IO 20 30

24 6 8 IO 12 14 16 18 20

NUMBER OF POPULATION DOUBLINGS

Chart 2. Kinetics of expression of the Al phenotype. A population of ~22 x106 cells in 11 flasks was synchronized (20); -8 x 106 cells served as theuntreated control population; -14 x 106 cells were treated with 21.5 jig of

propane sultone per ml for 14 hr after the cells entered S phase. The survivingpopulation (~28%) as well as the untreated cells were trypsinized, diluted 1:2 insMEM, replated, and kept in exponential growth by continuous subculturing insMEM as required. On each of the days indicated in parentheses, 2 to 5 x 10"

cells were trypsinized, pooled, and counted to determine the number of population doublings which had occurred. For each determination, a minimum of 106cells were assayed for Al growth (5x10" cells/dish), and the rest were replated

for continued propagation and subsequent assay.

concentrations, and after 14 hr, assayed for the percentage ofsurvival and plated at expression densities in sMEM. Theuntreated and the treated populations were maintained in exponential growth for 5 to 6 population doublings and thenassayed for frequency of TG-resistant colonies as described.The background frequency of TG-resistant cells was ~5 x1CT6. A dose-dependent increase in the frequency of TG-

resistant mutants was induced in the treated populations whichwas ~20-fold lower than the frequency of Al colonies seen

when such cells were propagated in sMEM and assayed inAgar1.

Effectiveness of the Synchronization Procedure of Miloand DiPaolo (20). Using flow cytometry techniques, we examined the degree of synchrony produced in the population bythe prescribed procedures (20). The results are shown in Chart4. After the arginine-deficient, glutamine-deficient MEM was

exchanged for sMEM, the DNA peak moved to the right indicating DMA synthesis (Chart 4, ÃŸand C) and then graduallyreturned to the original location (Chart 4D), indicating that,after 24 hr, cells that had been replicating their DNA hadcompleted mitosis and were again in the G, state. The profilesshown in Chart 4, B and C, suggested that there was asubpopulation of cells which did not participate in the synchronized movement through the cell cycle and may, in fact, havebeen composed of nonviable cells. To test this prediction, wecompared the cloning ability of cells which had been synchronized by this method with parallel cultures of cells which werekept to exponential growth. The results demonstrated that thecloning efficiency of the cells given the synchronization regimewas ~50% that of cells in exponential growth. The latter had acloning efficiency of 45%. We concluded that, although themethod gave a high degree of synchrony of DNA synthesisamong one cell subpopulation, a second subpopulation consisting of â€”50%of the cells was reproductively dead as a result

of the synchronization technique used (see also Table 1, Column 2).

Chart 3. The cytotoxicity, mutagenicity, and ability of various concentrationsof propane sultone to induce Al growth. Cells were synchronized (20) andexposed to the carcinogen just after the onset of S phase. After 14 hr, an aliquotwas assayed for initial cytotoxicity by measuring the percentage of survival of thecloning efficiency, and the rest was subcultured in sMEM. The cells were assayedas described for resistance to TG after 5 to 6 population doublings. They wereassayed as described for Al growth after 10 to 11 doublings.

20

IS

KI 10O

-i 5

Oer ,cLUI3CD

1 'O

0 HR

I 8 HR 24 HR

RELATIVE DNA CONTENT

Chart 4. The relative DNA content/cell as assayed by flow cytometry for cellssynchronized by the protocol of Milo and DiPaolo (20). Cells in exponentialgrowth in sMEM were refed with MEM lacking arginine and glutamine andsupplemented with 10% dialyzed FCS. After 24 hr, the medium was changed tosMEM containing 1 fig of anthralin per ml, and the cells were fixed for flowcytometry as indicated. A. DNA profile at time of change to sMEM; 6, DNA profile10 hr after A (the usual time of carcinogen treatment); C, DNA profile 18 hr afterA; D, DNA profile 24 hr after A.





Contribution of Anthralin. It was our understanding that thepurpose of pretreatment of the synchronized cells with anthralinprior to exposure to the mutagen/carcinogen was to enhancethe synchrony in some way.6 We, therefore, examined the

effect of anthralin on the rate of DNA synthesis in synchronizedcells using flow cytometry techniques. No differences could bedetected between cells treated with anthralin and controls nottreated with anthralin (data not shown). To test whether anthralin had some other mode of action which ultimately influenced the frequency of induction of Al colonies, we synchronized cells (passage II) and gave one set anthralin treatmentbut not the other set. Both sets were exposed to propane sultone (21 jug/ml) as described, and the progeny of eachwas assayed for Al colonies after 8 and 11 population doublings. The frequencies of Al colonies seen were identical,namely, ~700 x 10~5, suggesting that anthralin treatment was

without measurable effect.Lack of Tumorigenicity of the Al Cells Induced by Pro

pane Sultone. Cells propagated from Al colonies isolated fromAgar 1, 2, and 3 were assayed for tumorigenicity as described.Although ~100 mice were given injections (1 to 10 x 106

cells/mouse), no tumors resulted. In a number of instances,nodules formed and later regressed. Injection of ~106 Hs835T

cells invariably yielded tumors which proved to be adenocar-

cinomas.

DISCUSSION

Our results suggest that acquisition of Al growth in humanfibroblasts occurs as the result of single mutational event. Thisconclusion is supported by the 50- to 200-fold increase in

frequency of cells with this phenotype after exposure to astrong mutagen and by the fact that the Al phenotype is astable characteristic of cells which acquire the property (Table1). Furthermore, the induction of Al growth occurs in cellsexposed to the same concentrations of mutagen necessary toinduce TG resistance, a well-studied mutational marker in these

cells. Also, the induction of the Al phenotype exhibits anexpression curve similar to that seen with other mutationalmarkers, such as TG resistance (11, 18). The fact that theobserved frequency of Al colonies decreases after 13 population doublings suggests that mutagen-induced Al cells are at a

selective disadvantage by comparison with normal cells; i.e.,they have a longer growth cycle under the conditions used forexpression. A similarly shaped expression surve has beenreported for Al mouse cells derived by mutagen treatment (3).

The difference in frequency between induction of TG resistance and Al growth shown in Chart 3 is about 20-fold. These

data were derived from 2 separate exposures of cells of propane sultone, once for the Al study and again for the TGresistance study. The similarity of the dose response is obvious,but a replot of the induction data in Chart 3, ÃŸand C, as afunction of the cell survival [degree of cell killing (Chart 3/4)]would further emphasize the correlation. The reason for the20-fold difference is not known. Since the cloning efficiencies

of the cells at the time of selection for TG resistance or Algrowth in Agar 1 were similar (35 to 40%), the differenceprobably does not lie in the physiological state of the cultures.If, as we suppose, the 2 phenotypes, Al growth and TG resistance, are the result of single mutational events, possible explanations for the higher frequency of Al growth might be that the

target gene is much larger than the gene for TG resistance, thegene for Al growth contains a hot spot, or recovery is muchmore complete in the case of Al growth. If Al growth is theresult of a mutational event in normal cells, one expects to find,as we have, a low but measureable background frequency ofAl cells in the untreated population. This was reported recentlyfor mouse cells (3). The background of Al cells of Milo andDiPaolo (20) was below the level of detection, but this was alsotrue for us when we followed their formula for soft agar. Thedata in Chart 2 suggest that another reason why the frequencies of propane sultone-induced Al growth in our hands are 5-to 25-fold higher than for these investigators is that they missedthe peak of the expression of this phenotype by assaying after-21 doublings (20).

A similar explanation, i.e., not assaying cultures during thepeak of the expression period, could account for the very lowfrequencies of Al cells reported recently by Sutherland ef al.(26) after multiple exposures of human skin fibroblasts of UVradiation. Cells were assayed 5 to 9 days after irradiation with10 or 20 J/sq m. The frequencies observed ranged from 10 to800 x 10~6 cell plates. These data are comparable to the low

frequencies we also observed at the beginning of the expression curve in the cultures assayed on Day 6 or 8 (see Chart 2,approximately 4 or 5.5 population doublings). Presumably, ifthey had allowed sufficient time for full expression, the frequencies would have been ~ 10-fold higher. Note that in Chart

2 we plot the kinetics of expression as a function of populationdoublings rather than number of days posttreatment. This isbecause in related studies we have observed that the numberof days required to attain a particular number of cell doublingsdepends on the rate of growth and reflects the amount of DNAdamage induced in the target cells [i.e., there is a dose-de

pendent lag (19)]. Lack of comparable expression periodsresulting from differences in rate of cell doubling may alsoaccount for their lowered frequencies of Al cells induced inolder-passage cells (26). Recently, Kakunaga ef al. (15) also

reported induction of Al growth in human cells at very lowfrequencies (1 to 20 x 106) using 4-nitroquinoline 1-oxide.

Since the earliest assay was carried out 21 days posttreatment,it is possible that this was after the peak of expression.

A very high correlation has been reported between expression of the Al phenotype and tumorigenicity in mouse (3, 5)and hamster cells (2). However, in mouse cells, Al growth isseen after an expression period of 2 to 8 days (5 to 16population doublings) following mutagen treatment (3)whereas, in Syrian hamster embryo cells, such Al growth isseen only after 32 to 75 population doublings following mutagen treatment (2). This long period before acquisition of thisphenotype makes it doubtful that the Al growth observed in thehamster cells is the direct result of the mutagen-carcinogen.Nevertheless, the more important finding is that, in both mouseand hamster cells, it is from the population of Al cells thattumors arise.

Cells derived from human tumors have been shown to becapable of Al growth, and the cells isolated from the 2 independent human fibrosarcomas we have obtained in athymicmice with carcinogen-treated human cells also exhibit this

property. [The origin of one was described here; the secondwas obtained by treating KD cells with MNNG and using thefoci formation protocol of Kakunaga (14) (data not shown).] Infact, this characteristic is so selective for tumor cells that it

MAY 1981 1625




forms the basis of a method developed to identify the antitumordrugs to which particular human tumor cells are sensitive (10).However, the cell fusion studies of Stanbridge and Wilkinson(24) demonstrate that, in human cell hybrids, Al growth is notequivalent to malignant cell transformation. Their results, together with those reported here, suggest that acquisition of theproperty of Al growth by human fibroblasts is necessary butnot sufficient for malignant transformation. We postulate thatone or more additional steps, whose nature is as yet unknown,are required for malignant transformation and that, on occasion, they occur in human fibroblasts which have acquiredanchorage independence during the extended period of proliferation. This idea fits well with the notion that chemical- andradiation-induced carcinogenesis is a multistep process.

Borek (4) and Kakunaga (13, 14) who monitored foci formation by human fibroblasts following radiation or carcinogentreatment have also reported a correlation between this endpoint and tumor formation. Borek (4) found that cells isolatedfrom 5 foci showed Al growth and that 3 of the 5 producedfibrosarcomas in athymic mice. Kakunaga (14) found that cellsisolated from foci induced by carcinogen treatment showed Algrowth and that 7 out of 7 injected into athymic mice gavetumors. His data indicate that expression of focus formationrequires -13 population doublings. The high frequency of

tumors reported by both Borek (4) and Kakunaga (13, 14) mayreflect the fact that cells capable of foci formation represent atumorigenic subpopulation of Al cells. We are presently studying this possibility.

From Table 1, it is clear that the efficiency of growth in agar,whether for cells which are progeny of carcinogen-treated cellsor not, is enhanced when the cells are isolated from agar,propagated, and reinoculated into agar. We attribute this enhancement to the enrichment for cells with this AI phenotypeby cycling cells through agar. When colonies of cells areisolated from agar with a Pasteur pipet, one necessarily transfers with the agar some cells that remained viable but wereunable to grow as colonies. We have found that these cellsgrow in the plastic culture dish along with the cells from the Alcolonies that have been isolated. By recycling the cells throughagar as early as possible, we presume that we select againstcells lacking the Al phenotype, reducing the number of theselatter cells in the population. It is obvious from Table 1 that thefrequency of Al cells assayed in Agar 2 and Agar 3 is notinfluenced by the dose of the carcinogen used or the length ofthe expression time (compare Columns 7 and 8 with Columns10 and 11). This is to be expected if one selects cells fromAgar 1 that have the Al growth property as a stable heritablecharacteristic. Our results differ from those reported recentlyby Kakunaga et al. (15) who found that Al growth was inducedby 4-nitroquinoline-1-oxide but reported that most cells that

formed colonies in agar did not retain the Al phenotype. Sincefew details were available in his report, one can only speculatethat the decrease in frequency they observed was the result ofthe Al cells being diluted out by normal cells during an extendedpropagation.

In summary, the data presented in this report indicate thatthe treatment of early-passage randomly proliferating foreskin-

derived fibroblasts with doses of carcinogen/mutagen thatallow between 10 and 90% survival (as judged by cloning)followed by proliferation of the progeny for 8 to 13 populationdoublings before selection of Al growth (the optimal time may

vary with carcinogen dose) is all that is required for diploidhuman cells to acquire this phenotype. We do not attribute ourability to produce Al growth of human cells to the fact thatpropane sultone was chosen as the mutagen since we haveobtained similar results with MNNG. T-test analysis of the 25%

enhancement of the frequency of Al cells for populations synchronized and treated in S phase or propagated in 9sMEMshowed it to be significant (p < 0.01). However, the findingthat 50% of the cells are killed by the synchrony treatmentsuggests that it be used with caution. We are currently examining the critical factors involved in the second step of thetransformation process, namely, the formation of fibrosarcomas in athymic mice, using the tumorigenic human cells weinduced with MNNG in this study.

ACKNOWLEDGMENTS

We thank R. Corner, L. Lommel, L. Milam, L. Rowan, and T. Van Noord whoassisted with various aspects of this research and Dr. George Milo of The OhioState University who was of great assistance in helping us to understand thedetails of the transformation assay.

ADDENDUM

We have recently been able routinely to obtain fibrosarcomas upon injectionof Al cells into sublethally irradiated athymic mice.

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1981;41:1620-1627. Cancer Res K. Charles Silinskas, Suzanne A. Kateley, John E. Tower, et al. Fibroblasts by Propane SultoneInduction of Anchorage-independent Growth in Human

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