[CANCER RESEARCH 39, 2718-2726, July 1979]0008-5472/79/0039-0000$02.O0

Different Responses to Drugs and Serum of Cells Transformed by Various

Means

Robert Dubrow,1 Veronica G. H. Riddle, and Arthur B. Pardee2

Division of Cell Growth and Regulation, Sidney Farber Cancer Institute, Boston, Massachusetts 02 115, and Department of Pharmacology, Harvard Medical School,Boston, Massachusetts 02115

transformed line cannot a priori be generalized to all types oftransformed lines. This is particularly important because of theincreasing evidence that most human cancers are caused bychemicals (23). Yet most comparisons have used virally transformed cells (e.g. , Refs. 26 and 37).

Many previous studies have demonstrated differences between untransformed and transformed fibnobbastswith respectto growth in medium containing low concentrations of serum(4, 11, 25, 28, 35, 44, 52, 59, 66) on in medium containingvarious drugs (10, 15, 17, 19, 40, 43, 49, 56, 62). For themost part, these were restricted to comparisons between anuntransformed line and one or a few transformed lines, usuallyof the same type (mostly DNA tumor virus-transformed cells).Growth without anchorage has been investigated with polyoma(DNA virus)-tnansformed BHK cells (32). It is suggested fromstudies mostly done with SV4O-transfonmed3T3 cells (55), asparticularly related to tumor-forming ability.

Recent data suggest that DNA virus transformation does notprovide a general model for studying the properties of transformed cells. For example, cessation of growth in G0 is usualfor crowded onserum-deprived untransformed cells. Based onstudies with DNA virus-transformed cells (4, 11, 35, 43, 44,59, 66), it has long been held that transformed cells do notcease growth in G0 under these conditions. However, abenzo(a)pyrene-transformed 3T3 line has been found to arrestin G0 with low serum (25). Two mouse embryo-derived, 3-methylcholanthrene-transfonmed lines (36), as well as a varietyof RNA virus-transformed lines (39), very recently were re

ported to arrest in G0 at high density. With respect to drugs,both cytochalasin B (40) and picolinic acid (15) arrested growthof DNA and RNA virus-transformed cells differently.

The purpose of the present investigation was to determine towhat extent the response to low serum and to drugs of a singletransformed line can be generalized to all transformed lines ofthat class and to what extent such differences obtained with aclass of transformed lines can be generalized to all types oftransformants. In addition, we wished to identify specific drugsfor which such generalizations could be made. We have examined 10 transformed cell lines (DNA and RNA tumor virus,chemically and spontaneously transformed) and their untransformed counterparts for their growth responses with low concentrations of serum and with 16 drugs.

MATERIALS AND METHODS

Drugs. 3-Acetylpynidine, antimycin A, 6-azaunidine, caffeine,chboroquine,oligomycin, ouabain, picolinic acid, notenone, andtetracaine were obtained from Sigma Chemical Co., St. Louis,Mo. Actinomycin D was obtained from Merck, Sharp, andDohme, Rahway, N. J. Sodium arsenate was obtained from J.T. Baker Chemical Co., Phillipsbung, N. J. Sodium azide was

2718 CANCERRESEARCHVOL. 39

ABSTRACT

The purpose of this work was to determine whether differenttransforming agents similarly modify the growth of cells asmeasured by ability to grow in media containing low concentrations of serum on drugs. The responses of BALB/3T3 andseven transformed derivatives (DNA virus, RNA virus, andchemical) and of several sets of other cells were studied.Growth of the BALB/3T3 series during 3 days in mediumcontaining low serum was a function of the mode of transfonmation. Flow microfluonimetry and autoradiography were usedto show that DNA virus-transformed lines were distributedaround the cell cycle, whereas RNA virus and chemicallytransformed cells had accumulated in G1.

The growth responses to the drugs notenone, antimycin A,azide, obigomycmn,arsenate, chloroquine, ouabain, tetnacaine,actinomycin D, bucanthone, 6-azaunidine, 3-acetybpynidine, pmcolinic acid, caffeine, methylglyoxal bis(guanylhydrazone), andstneptovitacin A (some of which have been previously shown toaffect some untransformed and transformed cells differently)were determined by the change in cell number after a 2-dayperiod. There was not a consistent response by all transformedlines. For no drug was every transformed line either more orless sensitive than was the corresponding untransformed line.The lines transformed by the same agent generally respondeddifferently to a single drug. However, growth responses toseveral drugs were characteristic for a class of transformants,although not unique to that class. This study demonstrates thatthe growth behavior of a single transformed line, and in particulan of a DNA-virus transformed line, cannot be taken to represent the behavior of all transformed lines on even of all linestransformed by the same agent.

INTRODUCTION

Tumor- and non-tumor-forming cells are often compared inculture, with the aims of finding differences in growth controlor response to drugs. To relate such studies to the problem ofcancer, it is important to exercise care in selecting the particulan cell lines to be used. Cells in tissue culture can be transformed ‘‘spontaneously'‘on by DNA tumor viruses, RNA tumorviruses, chemicals, or radiation. Relatively little work has goneinto examining properties of transformed cell lines as a functionof their mode of transformation (15, 33, 34, 39, 40, 63).Differences between an untransformed line and one type of

1 Present address: Interdisciplinary Programs in Health, Harvard School of

Public Health, 665 Huntington Avenue, Boston, Mass. 021 15.2 Recipient of USPHS Grant CA 1 9949 To whom requests for reprints should

be addressed, at Sidney Farber Cancer Institute, 44 Binney Street, Boston,Mass. 02115.

Received November 16, 1978; accepted April 10, 1979.

on May 25, 2018. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Cell lineTransformingagentDesignation8Ref.BALB/3T3

mouse fibroblastsA31-CL7NoneA312Sv-A31

-CL29Sv4oSv-A31-2953Sv-A31-CL34SV4OSv-A31 -3452,53K-A31

-CL234Kirsten murine sarcomavirusK-A3157,

63M-A31-CL71Moloney

murine earcoma virusM-A311

,63B77-A31-CL1B77-Aous

sarcoma virusA-A3158,

63BP3T3CL7-5Benzo(a)pyreneBP-A3141BALB-3T3-D7,1

2-Dimethylbenz(a)anthraceneD-A3141Primary

mouseembryoNoneMEF47fibroblasts(C57BL/6)Swiss

3T3 mouse fibroblasts3T3None3T364SV3T3SV4OSV3T365Syrian

babyhamsterkidneyBHK21/CI13NoneBHK60PyBHK

(Ji )Polyoma virusPyBHK1 0,11Chinese

hamsterembryofibroblastsCHEF

18-1NoneCHEF-lB-i51CHEF16-2SpontaneousCHEF-i6-251a

Designation used in this paper.

Response of Transformed Cells to Drugs and Serum

obtained from Matheson, Coleman, & Bell, Nonwood, Ohio.Methylglyoxal bis(guanylhydnazone) dihydrochbonide monohydrate was obtained from Aldrich Chemical Co., Milwaukee,Wis. Lucanthone was obtained from George H. Hitchings, TheWeblcome Research Laboratories, Burroughs Wellcome andCo., Research Triangle Park, N. C. Streptovitacin A was obtamed from Dr. B. K. Bhuyan, The Upjohn Co., Kalamazoo,Mich.

Cell Culture. The cell lines used in this study are presentedin Table 1. A31-CL7, M-A31-CL71 , and B77-A31 -CL1 wereobtained from Dr. George Todaro, National Cancer Institute,Bethesda, Md. SV-A31-CL29, SV-A31-CL34, and K-A31-CL234 were obtained from Dr. Charles Schen, Sidney FarberCancer Institute, Boston, Mass. BP-3T3-CL7-5 was obtainedfrom Dr. Robert Holley, Salk Institute for Biological Studies,San Diego, Calif. BALB-3T3-D was obtained from Dr. RobertWeinberg, Massachusetts Institute of Technology, Cambridge,Mass. 3T3 and SV3T3 were obtained from Dr. Howard Green,Massachusetts Institute ofTechnology, Cambridge, Mass. BHKwas obtained from the American Tissue Culture Collection,Rockville, Md. PyBHK was obtained from Dr. Michael Stoker,Imperial Cancer Research Fund Laboratories, London, England. CHEF 18-1 and CHEF 16-2 were obtained from Dr. RuthSager, Sidney Farber Cancer Institute, Boston, Mass. Primarymouse embryo fibroblasts were prepared from late-termC57BL/6 embryos from which the head and internal organshad been removed (47).

Cells were routinely grown at 37°in a water-saturated 10%CO2-90% air atmosphere in Dulbecco's modification of Eagle'smedium (Gibco H21; high glucose) supplemented with 10%calf serum, penicillin (100 units/mI), and streptomycin (100

Table 1Cell lines

zg/mI) with the exception of CHEF 18-1 and CHEF 16-2, whichwere grown in Eagle's minimal essential a medium withoutnibonucleosides or deoxynibonucleosides (Gibco) modified toa (3.7-g/liter) concentration of sodium bicarbonate and supplemented with 10% fetal calf serum, penicillin, and streptomycin.

All of the transformed lines, with the exception of SV-A31-34, form tumors in animals. SV-A31-34 is a ‘‘flat,‘‘T-antigenpositive, non-tumor-forming transfonmant (52).

All cell lines were determined to be pleunopneumonia-Iikeorganism free, based on the ratio of [3H]unidine to [3H]unacibincorporation into RNA (54).

Flow Microfluorimetry. Cells were prepared for flow microfluonimetry as described by Yen and Pardee (69). Cells grownin 100-mm culture dishes were trypsinized and suspended in5 ml of medium. One ml was removed into a vial containingHanks' balanced salt solution with 0.5% formaldehyde for acell count (Coulten counter), and the remaining 4 ml werecombined with 4 ml from a duplicate plate and processed forflow microfluonimetny (Biophysics Systems CytofluorografModel 4800A).

Autoradiography. Autoradiography was performed using amodification of the method of Hambin(21). Cells were grown in35-mm culture dishes. To determine the fraction of cells synthesizing DNA, the cells were pulsed for a 1-hr period with[3H]thymidine (10 @zCi/mb)(40 to 60 Ci/mmol; New EnglandNuclear, Boston, Mass.). Following fixing and drying, emulsionwas put on the dishes, which were developed after 3 days.

Measurement of Response of Cell Lines to Drugs. Cellnumber was used to measure the effect of drugs on cell growth.Replicate cultures (1O@to 2 x 1O@cells per 35-mm culturedish) of the cell lines to be compared were established usingcells from log-phase cultures. On the following day, duplicatedishes were taken for cell counts (zero time point), and theculture medium from the remaining dishes was replaced withthe appropriate drug-containing medium (in duplicate). Duplicate control cultures received medium without drug. To minimize variability, drug-containing medium was prepared by diluting a sterile stock solution of the drug into enough mediumfor all of the cell lines being compared in that experiment.Ethanol and dimethyl sulfoxide were used as solvents for someof the drugs. The final concentration of ethanol on dimethylsulfoxide in the drug-containing medium did not exceed 0.25%,a concentration which for either of these solvents alone had noeffect on cell growth. Forty-eight to 52 hr after the addition ofdrugs, cell counts were performed. In control cultures, therewas a substantial increase in cell number after this time period(e.g., 9-fold for a typical 16-hr generation time), thus givingadequate time for differences to emerge. Furthermore, assayswere made at 2 days, since from the data obtained with bowserum, this was the time at which major differences were mostevident.

The effect of a drug on a given cell line was expressed asthe relative growth, defined as

RG3= (I@ @/I@ @)@@

where C0 @5cell count with drug, C is cell count in control, and

3 The abbreviation used is: RG, relative growth.

JULY 1979 2719

on May 25, 2018. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

R. Dubrow et a!.

Coi5 cell count at zero time. The use of this quantity normalizesgrowth rates in the presence of drug with respect to differencesin control growth rates among the cell lines. Preliminary expeniments indicated that under these experimental conditions cellgrowth was exponential in control cultures oven the course ofthe experiment. For cells that grew at an exponential rate inthe presence of drug, RG represents the ratio of the first-orderrate constant in the presence of drug to the first-order rateconstant in the control expressed as a percentage. A negativevalue for RG indicates cell loss.

Duplicate values for RG were calculated by randomly usingone and then the other of the duplicates for C0, C0, and C.Mean ± S.D. was determined using these values. For anexperiment performed only once, the S.D. represents intnaexperiment variability. For an experiment performed more thanonce, the S.D. represents a measure of both intna- and interexperiment variability.

Cell Counts. Following aspiration of the medium, the 35-mmculture dishes were rinsed once with 1 ml of trypsin (0.05%)-EDTA (0.5 mM) (in 138 mr,i NaCI-2.67 m@ KCI-1.47 mr@iKH2PO4-8.10 mM Na2PO4)solution and then incubated with 1ml of trypsmn-EDTAat 37°until the cells detached from theplate (generally 2 to 5 mm). The cell suspension was tnitunateduntil a single-cell suspension was obtained. This was placedinto a vial containing 9 ml of Hanks' balanced salt solution. Cellcounts were performed using a Coulten counter.

RESULTS AND DISCUSSION

Response of Transformed Cells to Growth In Low SerumConcentrations. When untransformed fibroblasts are placed inmedium containing a low concentration of serum, DNA synthesis ceases, and the cells become arrested in G0. It is now wellestablished that fibroblasts transformed by the DNA-containingviruses SV4O on polyoma continue to synthesize DNA and aredistributed through the cell cycle when grown with low serum(4, 11, 35, 44, 66). Their growth rate is slowed, and theyrapidly lose viability (43, 44).

There have also been reports of transformed lines which doenter a quiescent state under conditions of serum restriction(3, 20, 25, 31). For example, a chemically transformed BALB/3T3 line (BP-A31 ) behaved in this manner, although its quantitative growth requirement for serum was reduced (25). Howeven, there has been no report of a cell line known to betransformed by a DNA tumor virus which enters a G0 state inlow serum. Even T-antigen-positive ‘‘nevertants'‘did not regainthis property (13, 66). This suggested to us that the behaviorof DNA tumor virus-transformed cells in low serum might bethe exception rather than the rule. We therefore examined theresponse of 7 A-31-derived transformed lines (2 DNA virus, 3RNA virus, and 2 chemical) to growth in 0.2, 0.5, and 10%serum oven a 3-day period. Cell counts and flow microfluonimetry were performed on each day. In 10% serum, all cell linesgrew at an exponential rate with generation times ranging from14 to 19 hr. Growth curves in low serum are shown in Chart 1.All of the cell lines grew approximately exponentially oven thefirst 2 days of growth in 0.5% serum with generation times of19 to 29 hr, except for A-31 and M-A31 , which grew significantly more slowly. In 0.2% serum, greater inhibition of growthwas observed, with A-31 , M-A31 , and SV-A31-29 showing themost marked inhibition.

Time (hr@Chart 1. Growth curves of A-31 and its transformed derivatives in low serum.

Replicate cultures (1o@cells/ 100-mm culture dish) of A-31 and each of itstransformed derivatives were established by plating cells from log-phase culturesinto medium containing 10% serum. On the following day, duplicate dishes fromeach call line were taken for call counts and flow microfluorimetry (Chart 2). Theculture medium on the remaining dishes was appropriately changed (0.2 or 0.5%

serum). On each of the next 3 days, cell counts and microfluorimetry (Chart 2)were performed. 0, A31; •,Sv-A31-29; & Sv-A31-34; A, K-A31; U, M-A3i;., R-A31;0, BP-A31;0, D-A31.

The flow microfluonimetnic analyses of the cell lines growingin 10% serum were all quite similar. A representative DNAhistogram is shown in Chart 2A. However, in low serum the 2SV4O-transfonmed lines were clearly different from the others(Chart 2). Even after 3 days, the fraction of these cells in S andG2remained high. In contrast, in the other lines, the fraction ofcells in S and G2 was greatly reduced, both in 0.5 and 0.2%serum (Chart 2). Virtually all of the cells in A-31 and M-A31had a G1 DNA content.

These results were confirmed by autoradiography in anexperiment in which the fraction of cells synthesizing DNA wasmeasured on successive days of growth in 0.2% serum (Table2). Three groupings were observed: A31 and M-A31 , in whichDNA synthesis decreased most sharply; K-A31 , R-A31 , BPA31 , and D-A31 , in which DNA synthesis decreased moregradually but almost as completely; and SV-A31-29 and SVA31-34, in which DNA synthesis remained high.

Thus, cells transformed by different means responded to lowserum differently. Of particular interest was the Moboneysancoma virus-transformed line M-A31 , the growth arrest of whichin G1 was almost indistinguishable from that of the untransformed A-31 line. The 2 other RNA virus-transformed lines (K

2720 CANCERRESEARCHVOL. 39

on May 25, 2018. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

J'LiJ@Lij1\@@@.L

CelllineDays

in 0.2 serum-containingMedium0123A31

Sv-A31-29Sv-A31-34K-A31M-A31R-A31BP-A31D-A3167a

7975708245737323

567449214748697

46603581628233

5130606

109a

Percentage of Iabeled nuclei.

C account for the slower response of BP-A3i to serum restriction,

and similar differences may be operating in the case of K-A31,R-A3i , and D-A31 . In fact, we have found that the accumulationof BP-A31 cells in G, was a function of the cell density at whichthe cultures were deprived of serum.4 This result is consistentwith the depletion of growth factors determining the rate ofgrowth arrest. Our results, along with those of others, indicatethat DNA tumor virus-transformed cells have completely lost aserum requirement for the initiation of DNA synthesis. Flat andstandard SV4Otransformants, both of which contain the SV4O-induced T-antigen, lose this requirement (28, 52, 53). Thissuggests that the T-antigen itself may replace the function ofa serum factor(s) and itself may initiate DNA synthesis (67). Itis noteworthy that DNA virus-transformed lines undergo ‘uncontrolled' ‘nuclear division in the presence of cytochalasin B,whereas RNA virus-transformed lines exhibit ‘‘controlled'‘nuclear division (40).

Effect of Drugs on Growth of Transformed Cells. Ourresults with low serum encouraged us to determine whetherdifferent transformed cell lines respond to various drugs indifferent ways. The following drugs were used: rotenone, antimycin A, azide, oligomycin, and arsenate, which are respiratoryinhibitors (22); chboroquine, a membrane-active agent whichinactivates lysosomal function (30) and inhibits DNA repair(1 8); ouabain, an inhibitor of Na@-K'@-ATPase(61 ); tetracaine,a membrane-active agent (42); actinomycin D and lucanthone,inhibitors of rRNA synthesis (14); 6-azauridine, which decreases pynimidine precursor pools for RNA and DNA synthesis(7); 3-acetylpynidine, a nicotinamide antagonist (9); picolinicacid, which is both a nicotinamide antagonist and a chelatingagent (15); caffeine, an inhibitor of cyclic adenosine 3':5'-monophosphate phosphodiesterase (61) and DNA repair (29);methylglyoxal bis(guanybhydnazone),an inhibitor of polyaminebiosynthesis (50); and streptovitacin A, a protein synthesisinhibitor related to cycloheximide (5, 43).

The increase in cell number over a 2-day period was determined in the presence of several concentrations of a drug.Results are not given for drug concentrations which preventeda doubling in the cell number of all the lines compared becausethey do not provide useful comparisons. The results are presented in Tables 3 and 4 and are qualitatively summarized inTable 5. The most notable finding was the lack of consistencyof drug response among transformed lines. For no drug wereall transformed lines either more or less sensitive than werethe corresponding untransformed lines. All lines transformedby a particular mode did not exhibit the same response formost drugs. A particular type of transformation did not exhibita drug response which was unique to that mode of transformation. Thus none of the 16 drugs tested could unambiguouslydistinguish between transformed and untransformed lines, norcould a drug distinguish among DNA virus, RNA virus, andchemically and ‘‘spontaneously'‘transformed lines.

Some trends in the data could warrant further investigation:reduced sensitivity of DNA virus-transformed lines to caffeineand actinomycin D; increased sensitivity of DNA virus-transformed lines to respiratory inhibitors (except arsenate, an uncoupling agent); increased sensitivity of RNA virus-transformedlines to caffeine and 6-azaunidine; decreased sensitivity ofchemically transformed lines to respiratory inhibitors. Reduced

4 R. Dubrow, ‘I. G. H. Riddle, and A. B. Pardee, unpublished data.

Response of Transformed Cells to Drugs and Serum

B

DNA Content

Chart 2. Flow microfluorimetric analyses of A-31 and its transformed derivetives. See Chart 1 for experimental details. DNAcontent corresponds to G1phase(left peak), S phase (troughs), and G2 phase (right peak). The DNA histogramsfor A-31 , K-A31 , M-A31 , R-A31, BP-A31, and D-A31 were essentially the sameafter growth in 0.2% serum for 3 days as in 0.5% serum. A, M-A31 , 10% serum,Day 0; B, A-31 , 0.5% serum, Day 3; C, Sv-A31-29, 0.5% serum, Day 3; 0, SvA31-29, 0.2% serum, Day 3; E, sv-A31-34, 0.5% serum, Day 3; F, Sv-A31-34,0.2% serum,Day 3; G, K-A31, 0.5% serum,Day 3; H, M-A31•0.5% serum,Day3; I, R-A31, 0.5% serum,Day3; J, BP-A31, 0.5% serum,Day3; K, D-A31, 0.5%serum, Day 3.

Table2Fraction of cells synthesizing DNA in 0.2% serum as a function of

time(A31and its transformedderivatives)Log-phasecultureswere platedat 2.5 x 1o@cells/35-mm culture

dish in mediumcontaining10%serum.Onthe followingday,duplicatedisheswerepulsedfora 1-hr periodwith[3H]thymidine,andtheculturemediumontheremainingdisheswaschangedto 0.2%serum.Oneachof the next 3 days, duplicate dishes were pulsed with [3H]thymidine.Autoradiographywasperformedasdescribedin “MaterialsandMethods.‘‘At beast400 nucleiwerecountedfor eachdetermination.

A31 and R-A31) and the 2 chemically transformed lines (BPA31 and D-A3i ) took longer to respond to serum restriction.However, by the third day these cells also accumulated in G1.In contrast, the 2 DNA virus-transformed lines remained distnibuted throughout the cell cycle.

BP-A3i has been previously shown to deplete serum factors,including a factor of the fibroblast growth factor type, from themedium at a slower rate than does A-31 (25). In addition, aslightly lower serum concentration was required to initiate DNAsynthesis in BP-A31 than in A-31 . These 2 differences probably

JULY 1979 2721

J@@\:@I

on May 25, 2018. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

A. Dubrow et al.

C')

0aa>

a>

aVVaE0InCa

VC

U-LU

0C

001C0(001

V

0

C)a

LU

C

00Ca>

aa

a;C0C

C

aaa

01

aa

C)

a)

C)

a

VC

a ‘@E Ca) NE U'

.@ 2

a@

a a@ 0

aa aa: _ C5)0

@ C)C@ =

V .@C @<ca @Z@C

.@ ao@a

.@ @“-0C°I-ca a2CO

@d. . CC-@V

.@ @‘),,, .@ C

V ‘H@ C@@ a a

.@ a E 0 0@@a)@ a

0@ Z <@a.Q Q@

aC)

EaC0

@u)

>

z

a

>

z0

VaE0aCaC

C')

C')

0.CO

C')

C')

C')

C?

C')

>U)

0)

C')

>C,)

La@LU

C')

C0

a

CaC)C00

01

0

E0'-@‘@‘‘-‘-C'),-,-@-@-@-0J@- 00'-@Ct) N0)@C')04141

414141444111+14141444141 ‘H414141+1+44141410)0)

CON.@CO@ (0(01')OON'- C')C')@CI)ON0)COCO

@-(0C')NC')CO C')C')U)CO N.C')NCl)

C')C') It)NN.U) Ct)C')@

‘-O'-NCOC') @‘N@C')‘-C')CO@-0),-1') 0NY-NC@)COOC'J+444+4+444+4

+1+1+14141+4+4+441+4+441+141414141414141CO(0000(00)

N.CON-CON.CON- Cl)NCON.0) @N(0C')(O@-'-@-NN.@C')C0Cfl@01(0

(0N.C() NC')'-O COCn@Ct)@-(O(0@-0)C')C'4C'4@C')'@@

@-v,-'-'-@-NCS)NNC')N'- @-N@-C')NO+4+4+4+4+4+441+4+44441+4+4+4+4+4444141 ‘H4441+441140)C')@O)@

@CO@N.Ct)C')CL) C')0N.@-0) (0@Ct)CO@N.00CO'- (00)04(01')'-(0(0

0N.@ C')(0

NC') LI)N.

(00)11)(01')'-‘-0@-'-C')@

@‘-0(00@0)CO@'-@(0,- @(0 C')C')00'-1444+41414+4

41+4144111+4+4+4+414+4+144 41+141144141(0LO@CO@N.

,-(O@N-L1)C')C') C')C')C)(0CO @C')N0)0C')O)'-CO'-(01') 11)0)040)'-

NN-CC) C')LI)

CON CO(0

O)@(O@‘N'

N@(0@@NN0C@JC')'-F'-.Cfl@-'-@COCOj-,- @(0,-(01')'-4141414144+4+144+44441414414414141+1+4 +14141414141COOLOC')'-C')

0)COCON.COLI)CO NN.0CO(O LI)@C')@CI)@-(DC')O)CON.COC')@I)CO@N-C)

(0N.Ct) NCO LI) (0LI)

(0(0N.COC')@‘-‘-

‘-‘- N‘- C')@@,-N,-CO‘-‘-NC')(0@@ 0+1+141+1 +1+1 41+441414141414141+111+1+1N(0@

04(0 0C')C') 0)N Cl)(0

04LI) @(00)(0(0CO0)0)N.@N.(00)0)r'.(0 N.LOLI),-

(0N-0@CON'-CO(0

@(00(011)CD(O'-C')CON0411) (0(0‘-(0,-,-414141+14141

4441114114+144+14441411414 444444414141(0NC')N-(OCOL()

0)‘-@ It) C') @@0)0)0N ‘-N CO0)(OC')'-OCt)N. Cl)@ N U) C')0)0)

(0CO C')0)

(0LI) @@0Lt)LI)C')v.,-

01')'-‘-0'i'@-‘-(0 ‘- 0COO)'-‘-C'@‘-(04141

41+1+144+4+1414111 41 1141414141+141+10)0)

O)@0)N-LI) 10(0(0N.CO0)@ 1') N-N‘-(0

0 ‘-101')@ C')(0@LI) 21N-

C')

@ @°C,@

(0W-@‘)C'@0)0)

@‘C')NN@NC')C')C')C')NO)CO@@'-'-@U)'-@N@'(O@'‘-‘-COC')@C00‘-Nj-'-N.@C')

@C')N414141414141414141+411+1414144+141+14141+41141+144+141+1+1O)NCONN.@-C')'-

(0CS)N.C0C')(0@C')(0(00)04 @‘@LI)NLO@CONO)0)C0@'C')@COc')Nr.-'@o)@ N.Q)C')C')@C')N. (0NL()C'@(ON.(0

C0@@-—.@

.@

E@@

@ 01E01@-C @I)C

@‘COLI) zC@,NC@)‘-ONCOEOOOoädd'-dóóz

@ CC

(0@,NN

N.CI)000)1―.E——

:&o@csE@ z(0@'@'@ E,-C')@fl0L C')o0oa.,_,_1@4000(000000004000—

E_‘@‘E@01―...C@‘0C

C()a.LnE0'-'-C'JCC

00'@@C0@-...--

0101

N@0000

2722 CANCER RESEARCH VOL. 39

..L @a)@@ 0@ .@s V <

.@ < C@@ U) .@@@@@ ‘2@@ .@@

a)C •@ a .—@@ C'— >.@ a >,@ C@ C•@3@ 4)5)@ C@ @“>@ o.@@ a a@@ a)@'a)

Iilh .@@ U@@ .@n@@@@@ @<< o@@@@@ 3(6@@ .@ p@@

@ z

on May 25, 2018. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Relative growtha

Concentration 3T3 SV3T3 BHK PyBHK CHEF 18-1 CHEF 16-2

Response of Transformed Cells to Drugs and Serum

Table 4Effect of Drugs on Growth of 3T3, SV3T3, BHK, PyBHK, CHEF 18-1, and CHEF 16-2

Respiratory inhibitorsRotenone

77 ± 13±3

41 ±2 47±1

42 ±1 46±3

55 ±2 57±147 ± 187 ± 1

29 ± 2

—2 ± 029 ± 2

20 ± 583 ± 2

89 ±027 ±2

54 ±1

53 ±0

82 ±174 ±297 ±0

74 ±1

34 ±152 ±5

29 ±478 ± 2

0.08 @g/ml66± 7b(2)c47±4(2)0.1

@ag/ml38± 642± 260± 677±10.4@g/mlAntimycin

A

Azide0.04

@ag/mlO.05@g/ml0.5 mM1mM47

3333±

9± 6± 14(2) (2)32

3662±

5± 4± 4(2) (2)40

36±

8

±8Oligomycin0.04@g/ml

0.O5@g/ml0.1 zg/ml40

30±5

± 1038 30±4

± 045±8Arsenate0.025

mM0.03mM0.1mM0.2mM72±468±168 30±

2± 847 29±1±1Membrane-activeagents

Chloroquine@@2.5 @iM5,@M

25MM75 ,iM55±

260± 670

47±0

± 478 53±2

±2Ouabain0.

15 mM0.2mM0.25 mM65 49±

2± 045 36±

2± 143± 854±4Tetracaine75

laM200MM75±

079±036±1137±1RNA

inhibitorsActinomycinD0.001 5 @g/ml

0.003zg/mI0.005@g/ml0.O075@g/ml45

252723±

3± 4

± 4± 484

676154±

3± 4

± 5± 443

22±1

± 470 36±2

±4Lucanthone

6-Azauridine1

.5 gzg/ml2@tg/ml2.5@g/ml0.01 mM0.02mM54

3641±

4± 5± 2(2)65

4358±

1± 13± 1(2)57

53±

8

± 746 49±

1

±1Nicotinamide

antagonists3-Acetylpyridine0.3

@g/ml0.75@tg/ml62±

874± 149±441±1Picolinic

acide1 m@1.5mM2mM63±

569± 428

22±1

±1335 34±2±1MiscellaneousCaffeine

Methylglyoxal

400 @g/ml60O@g/ml

1 @M1

4

80±

10

± 5(2)69 56±

1

± 5(2)664456±

5± 7± 183

7045±

1±2±4bis(guanylhydra

zone)2.5@aM

5@tM

10 @LM5857±4± 245 40±4±117±621±4Streptovitacin

A0.02 gig/mI0.03@ig/mI0.04 @ag/ml44

37±8

± 10(2)50 54±6

± 2(2)43 30±5

±4(2)Controlgeneration

timer (hr)18.5±1.6(4)15.3± 2.7(4)12.9± 1.6(4)

9 ±3 36±164 ±1 81 ±1

32 ±2 31±1

25 ±10 (2) 75 ±8 (2)

30 ±1 44 ±1

25 ±10 (2) 61 ±3 (2)

28±327±428±412±157±243

11.8±3± 2.2(2)(4)29

± 0 5328 ±4 4212.4± 1.1(3) 11.3±1

±3± 0.6 (3)

a Defined in ‘‘Materials and Methods.―b Mean ±S.D.C Numbers in parentheses, number of separate experiments with duplicate cultures, if greater than one.

d Also an inhibitor of DNA repair.eAlsoachelatingagent., DeterminedfromC0and C(cellcountsat 0 and48 to 52 hr).

JULY 1979 2723

sensitivity of DNA virus-transformed lines to caffeine (43) and sensitive to a particular drug (e.g. , caffeine, azide, or 3-acetylactinomycin D (33, 34, 62, 68) has been observed previously, pynidine) while other transformed lines were less sensitive towhereas chemically transformed lines had the same sensitivity that drug than were the corresponding untransformed lines.to actinomycin D as dmdthe untransformed parental Imne(33, The response of transformed Imnesto tetnacaine, picolinic acid,34). SV-A31-29 was notable for its great sensmtivmtyto nespina- and methybglyoxal bis(guanylhydrazone) dmdnot much differtony inhibitors. Generally, some transformed lines were more from the response of the correspondmng untransformed lines.

on May 25, 2018. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

confidence. the designation was0.DrugNA

virusRNA virusChemical

BP-A31 D-A31“Spontaneous―CHEF

16-2Primary

untransformed

MEF5V3T3PYBHKSv-A31-29sv-A31-34K-A31M-A31R-A31RespiratoryinhibitorsRotenone—0—

— —— —00000+ +++AntimycinA—0— — ———000+000Azide++0———0+00++++0++Oligomycin00—

— —— —000++++0Arsenate0—++++000++—++++++Membrane-active

agentsChloroguine@'00— — —00000— —+ + +— ——Ouabain—0—00—000++++Tetracaine000NDC0—+000—

——RNA

inhibitorsActinomycinD+ + ++ +++ ++—00—+ ++—Lucanthone00—

—00000—+06-Azauridine+00+

+0— —— ——0000Nicotinamide

antagonists3-Acetylpyridine

Picolinicacid@@0 00 +—

— —

00 ND0 00 00 00 0+ 0++ +++

+———MiscellaneousCaffeine+++++0+++0—————+++——+++———Methylglyoxalbis(guanylhydrazone)—000000—0—0StreptovitacinA++——00——00—++0

R. Dubrow et a!.

Table 5

Relativegrowth oftransformedlinescomparedwithparentuntransformedlineExplanation of symbols. For at least one of the tested concentrations of the drug. the relative growth of the transformed line was: 0. 80 to 125%; + , 125 to 1S0%;

++, 150 to 200%; +++, more than 200%; —,66.7 to 80%; ——,50 to 66.7%; ———,less than 50% of the corresponding untransformed line. In addition to theabove criterion, data were grouped into the 4'4-, + + + . ——, and ———categories only when the difference between the relative growth of the transformed anduntransformed line for the drug was significant at a confidence level of at least 99%. Similarly. data were grouped into the + and —categories only when thedifference was significant at a confidence level of at least 95%. Significance was measured by the modified Student's t test. An analysis of covarlance was alsoperformed for comparisons in which more than one drug concentration was used.

In the cases where the first criterion for + + , + + + , ——, or ———was met, but the transformed line differed from the untransformed lines with 95% but not 99%confidence for that drug, the designation was dropped down to + or —. For all cases, it the transformed and untransformed lines differed with less than 95%

a Defined in “Materials and Methods.b Also an inhibitor of DNA repair.C ND, not done.

d Also a chelating agent.

A wide variability was noted in the frequency with whichtransformed lines differed from untransformed lines in drugsensitivity. Thus, the response of K-A3i differed from that ofA-3i in the case of only 2 drugs, and even for those 2 drugs,the differences were less than 50%. On the other hand, theresponse of SV-A31-29 differed from A-3i for 11 drugs, 9 ofthe differences being greaten than 50%. Some transformedlines, such as SV-A31-29, were generally more sensitive todrugs than were the corresponding untransformed lines, andother transformed lines, such as CHEF-i 6-2, were generallyless sensitive. Clearly, SV4O-transformed lines are not generalmodels for transformed cells.

The response of MEF to drugs was compared with that of A-31 to determine whether primary cells are generally more onless sensitive to drugs than established cell lines. No generalized difference was found, but the 2 lines had strikingly differentresponses to several drugs. This suggests that there may beimportant metabolic differences between primary cells andestablished cell lines. On the other hand, the differences maybe due to a trivial reason such as the 2 lines originating fromdifferent strains of mice or consisting of different cell types.

That drugs which have the same general metabolic effect oncells (e.g. , respiratory inhibitors and rRNA synthesis inhibitors)did not all affect the relative growth of a cell line in the same

way was not surprising. Such differences could be due to

secondary modes of action of drugs or to differences in exactpoints of action ondrug uptake (68).

The distribution of cells in and out of the cycle can also affectdrug sensitivity (6, 38, 43, 49), as is well documented forantineoplastic agents that act, for instance, in S on M phase.Some of the drugs used in this study are, no doubt, cyclespecific. However, cell cycle distribution is not likely to be amajor factor in the present study, since doubling times werenot very different for cells in a set (Tables 3 and 4).

Points at which drugs arrest cells in their cycle were notstudied as was done after serum deprivation. One would anticipate that some drugs would arrest cells specifically, as wasreported earlier for G1 arrest of BHK but not PyBHK cells bystreptovitacin A (43).

We can consider these results in connection with growthcontrol and chemotherapy of human tumors. As is too wellknown, human tumors differ one from another in many properties including growth rate and sensitivities to antineoplasticagents. Properties of tumor lines in culture also differ from oneto the other, as particularly studied with mouse lines (8). Thereare a variety of causes for such differences; these have notbeen well distinguished (45). Assuming a cbonalorigin of cancer(1 6, 27), the cell that was originally transformed could arisefrom different cell or tissue types or at various stages ofdifferentiation. Some of the properties of the original cell could

CANCERRESEARCHVOL. 392724

on May 25, 2018. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Response of Transformed Cells to Drugs and Serum

persist in the tumor cell. Secondary changes could anise insome cells of a tumor as it grows and its cells are located indifferent environments (45). Parts of one tumor could thusdifferently develop properties of growth (12) and drug sensitivity (24), as has been recently reported.

The source of differences between tumors particularly discussed here is due to various transforming agents. Even starting with the same untransformed cell, different agents canproduce cells with different properties. Several reports showthat spontaneously appearing tumor cells have a considerabledegree of growth control and that viruses or chemical cancinogens further change their properties (46). Variously modifiedcell types can anise even after a single agent transforms onekind of cell, as after infection of 3T3 cells with SV4Ovirus (48).

Thus, it seems hard to imagine that all tumors will have someuniversal, characteristic property such as response to a drug;indeed, no such common property has been found. To provideevidence of a change universal for all tumor cells from studies

of cells transformed by one agent such as SV4O virus appearsimpossible. The search for the most general traits of tumorforming cells should be carried out with a variety of cells thatare most closely related to cells of naturally occurring neoplasms.

ACKNOWLEDGMENTS

We thank David Karasic and Darless Lehtomaki for excellent technical assistance, Gordon Flowerdew and Kenneth Stanley for help with the statistics, andDavid Schneider for help in preparing the paper for publication.

REFERENCES

1 . Aaronson, S. A., and Rowe, W. P. Nonproducer clones of murine sarcoma

virus transformed BALB/3T3 cells. Virology, 42: 9—19, 1970.2. Aaronson, S. A., and Todaro, G. J. Developmentof 3T3-like lines from

BALB/c mouseembryocultures:transformationsusceptibilityto SV4O. J.Cell. Physlol.,72: 141—148,1968.

3. Baker, M. E. Stimulation of DNA synthesis by serum and amino acids in a ratneuroblastoma cell line. Exp. Cell Res.. 95: 121—126, 1975.

4. Bartholomew,J. C., Yokota, H.. and Ross,P. Effectof serumon the growthof BALB/3T3 A31 mouse fibroblasts and an SV4O-transformed derivative.J. Cell. Physiol.,88: 277—286,1976.

5. Bhuyan, B. K., and Fraser, T. J. Antagonism between DNA synthesisinhibitors and protein synthesis inhibitors in mammaliancell cultures. CancerRes., 34: 778-782, 1974.

6. Bradley, M. 0., Kohn, K. W., Sharkey, N. A., and Ewig, R. A. Differentialcytotoxicity between transformed and normal humancells with combinationsof amlnonucleoside and hydroxyurea. Cancer Res., 37: 2126—2131, 1977.

7. Burki.H. R. DNAcontentof mastocytomacellsincell cultureafter inhibitionof RNA synthesis with 6-azauridine. Cancer Res., 31: 1188—1191, 1971.

8. Butel, J. S., Dudley, J. P., and Medina, D. Comparison of the growthproperties in vitro and transplantability of continuous mouse mammarytumorcell lines and clonal derivatives. Cancer Res., 37: 1892—1900, 1977.

9. Caplan, A. I.. and Rosenberg, M. J. Interrelationship between poly (ADPRib) synthesis, intracellular NAD levels, and muscle or cartilage differentiatlon from mesodermal cells of embryonic chick limb. Proc. Nati. Acad. Sci.U. S. A., 72: 1852—1857,1975.

10. Clarke, G. D.. Shearer, M., and Ryan, P. J. Association of polyanionresistance with tumorigenicity and other properties in BHK/21 cells. Nature(Lond.), 252: 501 -503, 1974.

11. Clarke, G. D., Stoker, M. G. P., Ludlow, A., and Thornton, M. RequIrementof serum for DNA synthesis in BHK2I cells: effects of density, suspension,and virus transformation. Nature (Lond.), 22 7: 798—801, 1970.

12. Dexter, D. L., Kowalski, H. M., Blazar, B. A., Fliglel, Z., Vogel, R., andHeppner. G. H. Heterogeneity of tumor cells from a single mouse mammarytumor. Cancer Res., 38: 3174-31 81 , 1978.

13. Dubrow, R., Pardee, A. B., and Pollack, R. 2-Amino-Isobutyric acid and 3-0-methyl-a-glucose transport in 3T3. Sv40-fransformed 3T3 and revertantcell lines.J. Cell. Physlol.,95: 203—212, 1978.

14. Epifanova, 0. I., Makarova, G. F., and Abuladze, M. K. A comparative studyof the effects of lucanthone (Miracil D) and actinomycin D on the Chinesehamster cells grown in culture. J. Cell. Physiol., 86: 261—268,1975.

15. Fernandez-PoI, J. A., Bono, V. H., and Johnson, G. S. Control of growth bypicolinic acid: differential response of normal and transformed cells. Proc.Natl. Acad. Sd. U. S. A.. 74: 2889-2893, 1977.

16. Fialkow, P. J. Clonal origin of human tumors. Biochim. Biophys. Acta, 458:283-321, 1976.

17. Fodge, D. W., and Rubin, H. Differential effects of glucocorticoids on DNAsynthesis in normal and virus-transformed chick embryo cells. Nature(Lond.), 257: 804—806,1975.

18. Gaudln, D., Gregg, R. S., and Yielding, K. L. Inhibition of DNA repairreplication by DNA binding drugs which sensitize cells to alkylating agentsand X-rays. Proc. Soc. Exp. Biol. Med., 141: 543—547,1972.

19. Gierthy, J. F., and Studzinski, G. P. Absence of aminonucleoside-sensitivesteps in the cell cycle of Sv40-transformed human fibroblasts. Cancer Res.,33: 2673-2676. 1973.

20. Gill, G. N.. and Weidman, E. R. Hormonal regulation of initiation of DNAsynthesis and of differentiated function in Y-1 adrenal cortical cells. J. Cell.Physlol., 92: 65—72,1977.

21. Hamlin, J. L. S phase synchrony in monolayer CHO cultures. Exp. Cell Res.,100: 265-275, 1976.

22. Harstein,W. G. Uncouplingof oxidativephosphorylatlon.Blochem.Blophys.Acta, 456: 129—148, 1976.

23. Heidelberger.C. Chemicalcarcinogenesis.Cancer (Phila.), 40: 430-433,1977.

24. Heppner, G. H., Dexter, D. L., DeNucci,T., Miller, F. R., and Calabresi,P.Heterogeneity in drug sensitivity among tumor cell subpopulations of a singlemammary tumor. Cancer Res., 38: 3758-3763, 1978.

25. Holley, R. W., Baldwin, J. H., Kiernan, J. A., and Messmer, T. 0. Control ofgrowth of benzo(a@yrene-transformed 3T3 cells. Proc. Natl. Acad. Sd. U.S. A., 73: 3229-3232, 1976.

26. Hynes, R. 0. Cell surface proteins and malignant transformation. Biochim.Biophys. Acts, 458: 73- 108. 1976.

27. lannaccone, P. M., Gardner, R. L., and Harris. H. The cellular origin ofchemically induced tumours. J. Cell Sd., 29: 249—269,1978.

28. Jainchill, J. L., and Todaro, G. J. Stimulation of cell growth in vitro by serumwith and without growth factor. Exp. Cell Res., 59: 137- 146, 1970.

29. Kihlman, B. A. Effects of caffeine on the genetic material. Mutat. Res., 26:53-71, 1974.

30. Lie, S. 0.. and Schofield, B. Inactivation of lysosomal function in normalcultured human fibroblasts by chloroquine. Biochem. Pharmacol., 22:3109-3114, 1973.

31 . Lindgren, A., and Westermark, B. Serum requirement and density dependentinhibition of human malignant glioma cells in culture. Exp. Cell Res., 104:293—299,1977.

32. MacPherson, I., and Stoker, M. Polyoma transformation of hamster cellclones. An investigation of genetic factors affecting cell competence. Virology, 23: 291-294, 1964.

33. Mallucci, L., Poste, G. H., and Wells, V. Synthesis of cell coat in normal andtransformed cells. Nat. New Biol., 235: 222—223,1972.

34. Mallucci, L., Wells, v., and Young, M. R. Effect of trypsin on cell volume andmass. Nat. New Biol., 239: 53-55, 1972.

35. Martin, R. G. and Stein, S. Resting state in normal and simian virus 40transformed Chinese hamster lung cells. Proc. NatI. Acad. Sd. U. S. A., 73:1655-1659, 1976.

36. Moses, H. L., Proper, J. A., volkenant, M. E., Wells, D. J., and Getz, M. J.Mechanism of growth arrest of chemically transformed cells in culture.Cancer Res., 38: 2807-281 2. 1978.

37. Nicolson, G. L. Trans-membrane control of the receptors on normal andtumor cells. II. Surface changes associated with transformation and malignancy. Biochim. Biophys. Acta, 458: 1—72,1976.

38. O'Neill, F. J. Selective destruction of cultured tumor cells with uncontrollednuclear division by cytochalasin B and cytosine arabinosade.Cancer Res.,35: 3111-3115, 1975.

39. O'Neill, F. J. Differential in vitro growth properties of cells transformed byDNA and RNA tumor viruses. Exp. Cell Res., 117: 393-401 . 1978.

40. O'Neill, F. J., Miller, T. H., Hoen. J., Stradley, B., and Devlahovich, v.Differential response to cytochalasanB among cells transformed by DNA andRNA tumor viruses. J. NatI. Cancer Inst., 55: 951—955,1975.

41 . Oshiro, Y., and Di Paolo, J. A. Changes in the uptake of 2-deoxy-D-glucosein BALB/3T3 cells chemically transformed in culture. J. Cell. Physlol., 83:193—202,1974.

42. Papahadjopoulos, D., Jacobson, K., Poste, G., and 5hepherd, G. Effects oflocal anestheticson membrane properties. I. Changes in the fluidity ofphospholipid bilayers. Biochim. Biophys. Acts, 394: 504—519, 1975.

43. Pardee.A. B., andJames.L. J. Selectivekillingof transformedbabyhamsterkidney (BHK) cells. Proc. Nafi. Acad. Sci. U. S. A., 72: 4994-4998, 1975.

44. Paul, D., Henahan, M., and Walter, S. Changes in growth control and growthrequirements associated with neoplastic transformation in vitro. J. NatI.Cancer Inst., 53: 1499—1503, 1974.

45, Ponten. J. The relationship between in vitro transformation and tumorformation in vivo. Biochim. Blophys. Acts, 458: 397—422,1976.

46. Rhim, J. S. Transformation of human calls in culture by N-methyl-N-nitro-Nnitrosoguanidine. Nature (Lond.), 256: 751—753,1975.

47. Riddle,v. G. H., and Harris, H. Synthesisof a liverenzymein hybridcells.

2725JULY 1979

on May 25, 2018. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

R. Dubrow et a!.

J. Cell Sci., 22: 199—215,1976.48. Rlsser, R., and Pollack, R. A non-selective analysis of SV4Otransformation

of mouse 3T3 cells. Virology, 59: 477-489, 1974.49. Rozengurt, E., and Po, C. C. Selective cytotoxicity for transformed 3T3

cells. Nature (Lond.), 261: 701—702,1976.50. Rupniak, H. T., and Paul, D. Lack of a correlation between polyamine

synthesis and DNA synthesis by cultured rat liver cells and fibroblasts. J.Cell. Physiol., 96: 261—264,1978.

51 . Sager, R., and Kovac, P. E. Genetic analysis of tumorigenesis: I. Expressionof tumor-forming ability in hamster hybrid cell lines. Somatic Cell Genet., 4:375—392,1978.

52. Scher, C. D., and Nelson-Rees, W. A. Direct isolation and characterizationof ‘‘flat'â€S̃V4O-transformed cells. Nat. New Biol., 233: 263—265, 1971.

53. Scher, C. D., Pledger, W. J., Martin, P., Antoniades, H., and Stiles, C. D.Transforming viruses directly reduce the cellular growth requirement for aplatelet derived growth factor. J. Cell. Physiol., 97: 371—380,1978.

54. Schneider, E. L., Stanbridge, E. J., and Epstein, C. J. Incorporation of ‘H-uridine and ‘H-uracilinto RNA. A simple technique for the detection ofmycoplasma contamination of cultured cells. Exp. Cell Res., 84: 311—318,1974.

55. Shin, S., Freedman, v. H., Risser, R., and Pollack, R. Tumorigenicity ofvirus-transformed cells in nude mice is correlated specifically with anchorageindependent growth in vitro. Proc. NatI. Acad. Sd. U. S. A., 72: 4435—4439,1975.

56. Shohan, J., Inbar, M., and Sachs, L. Differential toxicity on normal andtransformed cells in vitro and inhibition of tumour development in vivo.Nature (Lond.), 227: 1244—1246,1970.

57. Stephenson, J. R., and Aaronson, S. A. Antigenic properties of murinesarcoma virus-transformed BALB/3T3 nonproducer cells. J. Exp. Med.,135: 503—515, 1972.

58. Stephenson, J. R.. Reynolds, R. K., and Aaronson, S. A. Characterization of

morphologic revertants of murine and avian sarcoma virus-transformed cells.J. Virol., 11: 218—222,1973.

59, Stoker, M. G. P. Tumour viruses and the sociology of fibroblasts. Proc. R.Soc. Lond. Biol. Sci., 181: 1—17,1972.

60. Stoker, M. G. P., and Macpherson, I. A. Syrian hamster fibroblast cell lineBHK21 and its derivatives. Nature (Lond.), 203: 1355—1357, 1964.

61 . Stryer, L. Biochemistry, pp. 775, 81 1. San Francisco: W. H. Freeman & Co.,1975.

62. Subak-Sharpe, H. Biochemically marked variants of the syrian hamsterfibroblast cell line BHK 21 and its derivatives. Exp. Cell Res., 38: 106—119,1965.

63. Todaro, G. J., DeLarco, J. E., and Cohen, S. Transformation by murine andfeline sarcoma viruses specifically blocks binding of epidermal growth factorto cells. Nature (Lond.), 264: 26—30,1976.

64. Todaro, G. J., and Green, H. Quantitative studies of the growth of mouseembryo cells in culture and their development into established lines. J. CellBioI., 17: 299-31 3, 1963.

65. Todaro, G. J., Green, H., and Goldberg, B. D. Transformation of propertiesof an established cell line by SV4Oand polyoma virus. Proc. Nail. Acad. Sci.U, S. A., 51: 66-73, 1964.

66. vogel, A., and Pollack, R. Isolation and characterization of revertant celllines. VII. DNA synthesis and mitotic rate of serum-sensitive revertants innon-permissive growth conditions. J. Cell. Physiol., 85: 151—162, 1975.

67. Weinberg, R. A. How does T-antigen transform cells? Cell, 11: 243—246,1977.

68. Williams, J. G., and Macpherson, I. A. The uptake of actinomycin D bynormal and virus transformed BHK21 hamster cells. Exp. Cell Res., 91:237—246,1975.

69. Yen, A., and Pardee. A. B. Arrested states produced by isoleucine deprivation and their relationship to the low-serum produced arrested state in Swiss3T3 cells. Exp. Cell Res., 114: 389—395,1978.

2726 CANCER RESEARCH VOL. 39

on May 25, 2018. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1979;39:2718-2726. Cancer Res   Robert Dubrow, Veronica G. H. Riddle and Arthur B. Pardee  by Various MeansDifferent Responses to Drugs and Serum of Cells Transformed

  Updated version

  http://cancerres.aacrjournals.org/content/39/7_Part_1/2718

Access the most recent version of this article at:

   

   

   

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

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

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

  Permissions

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

.http://cancerres.aacrjournals.org/content/39/7_Part_1/2718To request permission to re-use all or part of this article, use this link

on May 25, 2018. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from