[CANCER RESEARCH 39, 3051 -3057, August 1979]0008-5472/79/0039-0000$02.00

Phenotypic Properties and Tumor Promoter-induced Alterations in RatEmbryo Cells Transformed by Adenovirus1

Paul B. Fisher,2 Neil I. Goldstein, and I. Bernard Weinstein

Cancer Center/Institute of Cancer Research and the Division of Environmental Science, Columbia University College of Physicians and Surgeons, New York, NewYork 10032 (P. B. F., I. B. W.j, and Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19 104 (N. I. G.J

irradiation (10, 35, 39), or UV (6) has been shown to result inenhancement of transformation by SV4O or adenovirus. Themechanism underlying this apparent synergism is not presentlyknown. It has been suggested that DNA damage and repairmay be important mediators of this enhancement (8). Hirai etal. (28) found that, under conditions in which the chemicalcarcinogen 4-nitroquinoline-i -oxide increased the frequencyof SV4Otransformation of Chinese hamster embryo cells, therewas increased SV4O nuclear penetration and an increase inthe amount of SV4O DNA integrated into total cellular DNA. Itwas not possible to ascertain, however, whether more SV4Ogenomes were integrated per cell or more cells in the infectedpopulation contained integrated genomes. Methyl methanesulfonate, pretreatment of Syrian hamster embryo cells, alsoresults in increased incorporation of simian adenovirus 7 DNAinto cellular DNA (15). The mechanism by which such combined treatments affect the transformation process has not,however, been analyzed in detail.

The importance of multiple factors in the carcinogenic process in the intact animal is clearly demonstrated by the 2-stagemouse skin assay in which application of an ‘‘initiatingagent―followed by repeated application of a ‘‘promotingagent' ‘resuits in multiple skin neoplasms (for review, see Ref. 2). Themost potent tumor-promoting agents on mouse skin are TPA3and related plant diterpenes (for reviews, see Refs. 4, 27, and41). TPA has been shown to induce a spectrum of changes incells in culture, some of which are also observed when thiscompound is applied to mouse skin (23, 42, 43). TPA-inducedalterations in cells in culture include changes in morphologyand growth(14, 42—44),altered cell surface properties(3, 19,34), enhanced 2-deoxyglucose uptake (1 6), and induction or

enhancement of piasminogen activator production (42—45).inmany cases, TPA-induced modifications in normal cells mimicthose normally associated with cells transformed by viruses orchemical carcinogens. The ability to study TPA-cellular interactions in tissue culture and the reversibility of some of theaction of TPA (44) makes this an ideal compoundto studythephenotypic changes associated with the transformed state.

in previous studies, we demonstrated that pretreatment of2°REcells with the chemical carcinogens BP or DMBA enhances the transformation induced by HStsl 25 (24). In addition, continued growth of solvent or carcinogen-pretreatedH5tsi 25-infected 2°REcultures in TPA resulted in a furtherenhancement in the yield of transformed foci when compared

3 The abbreviations used are: TPA, I 2.O-tetradecanoylphorbol-1 3-acetate;

2°RE, secondary rat embryo; BP, benzo(a)pyrene; DMBA, 7,12-dlmethylbenz(a)anthracene; HStsl 25, human adenovlrus type 5 temperaturesensItive mutant; SD, saturation density; RE, rat embryo; LETS, large externaltransformation-sensitive protein; DMEM, Dulbecco's modified Eagle's minimumessential medium; FBS. fetal bovine serum; low-Ca2@medium, 0.1 m@Ca2@Dulbecco's modified Eagle's minimum essential medium; PBS, phosphatebuffered salIne (0.01 PAphosphate buffer and 0. 15 MNaCI, pH 7.4); SDS, sodiumdodecyl sulfate; Con A, concanavalin A.

AUGUST1979 3051

ABSTRACT

Seven clones of secondary rat embryo cells transformed bya temperature-sensitive mutant of human adenovirus type 5were analyzed to determine whether transformants obtainedfrom cultures treated with chemical carcinogen prior to virusinfection had a different phenotype than those obtained fromcultures treated with virus alone. In addition, the effects ofprolonged serial passage and the effects of exposure of theclones to i 2-O-tetradecanoylphorbol-i 3-acetate on theexpression of several markers of transformation were alsodetermined. When compared to normal secondary rat embryocells, most of the transformants had a reduced populationdoubling time, increased saturation density, reduced serumrequirement, increased plasminogen activator production, reduced large external transformation-sensitive protein, increased lectin agglutinability, and decreased anchorage dependence (i.e., growth in agarose and agar). Prolonged serialpassage of the transformants led to a spontaneous increase incloning efficiency in agar. There was no consistent differencebetween the phenotypes of transformants obtained from cultures treated with carcinogen plus virus or those of transformants obtained with virus alone, although clones obtained fromthe carcinogen-pius-virus treatment appeared to express anchorage independence at earlier subpassages. The most striking finding was that 12-O-tetradecanoylphorbol-1 3-acetatecaused an appreciable enhancement of the growth in agar ofall of the transformants. Two early-passage-transformed clonesgrew in agar only in the presence of i 2-O-tetradecanoylphorbol-i 3-acetate. The tumor promoter also increased the saturation densities and enhanced the cloning efficiencies in liquidmedium of most of the transformants. These effects were notas striking with normal secondary rat embryo cells.

These results suggest that following adenovirus transformation there is a spontaneous progression in the expression ofmarkers of transformation and that the phorbol ester tumorpromoters can accelerate this process.

INTRODUCTION

Since interaction between multiple factors may be importantin the causation of specific human cancers, it is of interest toexamine such interactions in model cell culture systems. Thetreatment of tissue culture cells with chemical carcinogens (6—8, 13, 24, 28), hormones (32), DNA base analogs (6, 40), X

I Research was supported by Contract NOl .CP-2-3234 from the NationalCancer Institute, Department of Health, Education and Welfare and Grant 5 F32CA 05640-02 from the National Cancer Institute.

2 To whom requests for reprints should be addressed, at Division of Environ

mental Sciences, Institute of Cancer Research, College of Physicians and Surgeons of ColumbIa University, 701 West 168th Street, New York, N.Y. 10032.

Received November 10, 1978: accepted May 3, 1979.

Research. on January 22, 2019. © 1979 American Association for Cancercancerres.aacrjournals.org Downloaded from

P. B. Fisher et aI.

to cultures not exposed to this tumor promoter (24). Underthese conditions, exposure of the normal cultures to carcinogen or TPA without virus infection did not produce the characteristic epithelioid foci seen with adenovirus transformation.H5tsl 25 is of particular interest because it has a higher transformation efficiency than does wild-type adenovirus 5, and yettransformation occurs at a temperature (37°)at which thismutant can not replicate (26, 31).

In the present paper, we have studied in considerable detailthe phenotypic properties of 7 morphologically transformedclones obtained from RE cultures infected with H5tsi 25 withand without prior exposure of the cultures to BP or DMBA.These clones were scored for several transformation-associated properties, i.e., SD, growth in low serum, anchorageindependence, plasminogen activator production, LETS, andlectin agglutinability, to determine to what extent the exposureto the polycyclic aromatic hydrocarbons had influenced thephenotypes of the transformants. We have also examined theeffects of prolonged serial passage of the cloned transformantsand the effect of exposing them to TPA on the expression ofcertain markers of transformation. A finding of considerableinterest is that, whereas normal 2°REcells did not grow inagar, either in the presence or absence of TPA, TPA markedlyenhanced the growth in agar of the adenovirus-transformedclones.

MATERIALS AND METHODS

Cells and Media. Primary RE cells were establishedfrom14-day-old Sprague-Dawley RE's as described by Fisher andGoldstein (20), using DMEM supplemented with i 0% FBS andpenicillin-streptomycin. H5tsi 25-transformed RE cells wereformed as previously described (24). Seven independentlyisolated H5tsi 25-transformed RE clones were obtained fromseparate transformation plates designated: Ad-Al 8-E and AdE7-E (early passage <1 0, solvent-treated controls), Ad-Ai 8-L

and Ad-E7-L (later passage >25, solvent-treated controls),early- and late-passage Bp-Ad-D2 and BP-Ad-Ei i (BP-pretreated cultures), and early- and late-passage DMBA-Ad-A6,DMBA-Ad-B8, and DMBA-Ad-C5 (DMBA-pretreated cultures)were utilized for further characterization. Cloned populationsof H5tsi 25-transformed RE cells were grown in Iow-Ca2'@'medium (5) supplemented with 7.5% FBS. The presence ofH5tsi 25 DNA sequences in all of these transformants wasconfirmed by hybridization of cellular DNA with restrictionenzyme-cleaved DNA fragments of 32P-labeledtype 5 adenovirus DNA as previously described (31). These data will bepublished separately.4 All cultures were grown at 37°in a 5%CO2-humidified air incubator.

Population-doublingTime and SD. The averagepopulationdoubling times and SD's of 2°REcells and H5tsi 25-transformed clones, in the presence or absence of TPA, wereobtained by plating 5 x 1O@ceils/50-mm tissue culture platein low-Ca2@medium at 37°,changing the medium after 6 or 24hr to low-Ca2'@medium with or without 100 ng of TPA per ml,and determining cell numbers over a 21-day period. Cells intriplicate plates were dispersed with trypsin-EDTA [0.25% trypsin (w/v) and 0.02% EDTA (w/v)] and counted every 24 hr,using a Model Z, Coulter counter. The medium was changed

4 K. Dorsch-Häsler, P. B. Fisher, I. B. Weinstein, and H. S. Ginsberg, manu

script In preparation.

every 72 hr with or without 100 ng of TPA per ml. Populationdoubling times were calculated over a 72-hr period corresponding to the logarithmic phase of growth (21). SD's represent the maximum cell densities obtained in confluent culturesgrown in the presence or absence of TPA. The values given forpopulation-doubling times (Table i ) represent the mean ±S.D.of triplicate determinations. The values for SD's (Chart 1) arethe means of triplicate determinations in which the variationwas less than 5%.

Growth in Low-Serum Medium. The ability of normal andadenovirus-transformed RE cells to grow in reduced amountsof FBS (1% as opposed to 7.5%) was assessed using theprotocol described above, except the medium added at 24 andevery 72 hr was low-Ca2@medium with 1% FBS, with or without100 ng of TPA per ml. Cell counts were determined every 24hr over a 21-day period. The ability of cells to undergo aminimum of 3 population doublings over a 1-week period wasconsidered as positive indication of growth in low serum.

Liquid Cloning Efficiency. One hundred or 200 H5tsi 25-transformed 2°REcells, or 1000 2°REcells, per plate, wereseeded into twenty 50-mm plates in low-Ca2@medium. Themedium was changed after 24 hr to Iow-Ca2@medium with orwithout 100 ng of TPA per ml, and thereafter 2 times/weekwith the same medium. Colonies of >50 cells were enumeratedafter 2 weeks of growth (24).

Agar and Agarose Cloning Efficiency. The ability of 2°REcells and HStsl 25 transformants to grow in an anchorageindependent manner, i.e., for single cells to give rise to spherical colonies without attachment to a solid substrate, wasassayed in either agar or agarose as described previously (i 8).in brief, 1O@,1O@,or 1O@cells in low-Ca2@medium supplemented with 0.4% Noble agar or Seakem agarose were layeredon 0.8% agar or agarose bases prepared in low-Ca2@medium.The plates were fed once a week with 3 ml of either 0.4% agaror agarose in low-Ca2@medium. Colonies >0. 1 mm werescored after 14 days, and colonies >0.2 mm were scored after21 days (37). The effect of TPA on growth in agar was determined as above except that 100 ng of TPA per ml was incorporated into both the base and the upper layers.

Plasminogen Activator Production. The amount of plasminogen activator produced by normal 2°REcells and HStsl 25-transformed RE cells was monitored from living cells by quantitating the amount of 125I-labeledpeptides released by digestion of iodinated fibrin (45). Two x 10@cells were seeded on5-cm-diameter fibrin-coated plates, containing 4 to 7 x 1O@cpm of 1251-fibrin(10 @zg/cm)in DMEM plus 5% FBS andallowed to attach for 4 hr. The cultures were then washed twicein PBS, and 5 ml of DMEM plus 5% FBS were added. Aliquotsof 400 ;.tlfrom triplicate samples were removed after 6 and 18hr, and the solubilized fibrin was determined by scintillationcounting (Nuclear-Chicago Corp., Des Plains, III.) in Aquasol(New England Nuclear, Boston, Mass.) using the 3H channel.Percentage of fibrin digested was determined from the ratio ofcpm released in the presence of sample (adjusted for fibrinreleased in the presence of serum but in the absence ofsample) to the total cpm released by trypsin.

LETS Determination-lodination and SDS-PolyacrylamideGel Electrophoresis. Five x i O@normalor adenovirus-transformed RE cells were plated in 25-sq cm T-flasks and incubatedfor 24 hr at 37°.The medium was removed, the monolayer waswashed 3 times with PBS, and i ml of PBS containing 5 mM

3052 CANCERRESEARCHVOL. 39

Research. on January 22, 2019. © 1979 American Association for Cancercancerres.aacrjournals.org Downloaded from

Growth and cloning properties of H5tsi 25-transformed RE cells in the presence or absence ofTPAPopulation-doubling

time (hr)― Liquid cloning efficiency (%)‘ +TPA/ Agar cloning efficiency (%)d +TPA/—TPA(liq- —TPA(agar

Cell type and designationa —TPA +TPA —TPA +TPA uid cloning) —TPA +TPAcloning)Normal2°RE

32.3 ±i.2e 31.6 ±1.5 3.i ±0.3 3.5 ±0.3 1.1 <0.OOi <0.0010Adenovirus-transformedAd-A18-E

32.5 ±1.0 29.5 ±1.0 i2.4 ±2.2 25.3 ±2.1 2.0 <0.001 0.1 ±0.0>100Ad-A18-L19.4 ±2.1 20.2 ±1.7 26.4 ±3.7 32.4 ±2.6 1.2 0.1 ±0.0 0.5 ±0.15.0Ad-E7-E46.3 ±3.5 45.7 ±2.8 4.7 ±0.4 7.2 ±0.7 1.5 <0.001 0.2 ±0.1>200Ad-E7-L21.4 ±1.7 20.2 ±2.1 12.4 ±i.i 17.3 ±1.6 i.4 1.2 ±0.1 2.7 ±0.32.3BP-pretreated,

adenovirustransformedBP-Ad-D2-E

22.8 ±0.9 20.1 ±0.5 22.6 ±2.3 36.2 ±i.9 1.6 0.2 ±0.0 0.5 ±0.22.5BP-Ad-D2-L.19.7 ±0.7 19.6 ±0.7 24.2 ±3.1 40.i ±2.6 1.7 4.1 ±0.3 7.1 ±0.51.7BP-Ad-E11-E18.2 ±1.0 19.1 ±0.6 33.2 ±2.9 51.5 ±4.i 1.6 1.1 ±0.3 2.9 ±0.32.6BP-Ad-E11-L15.2 ±0.5 15.4 ±0.7 35.6 ±3.0 49.6 ±3.4 1.4 3.0 ±0.8 11.6 ±0.93.9DMBA-pretreated.

adenovirustransformedDMBA-Ad-A6-E

22.7 ±1.5 i9.6 ±0.6 42.2 ±3.5 64.5 ±3.8 1.5 3.9 ±0.8 6.5 ±1.21.7DMBA-Ad-A6-L20.8 ±1.7 18.4 ±1.2 40.i ±2.6 70.3 ±2.9 1.8 6.7 ±0.7 10.7 ±1.31.6DMBA-Ad-B8-E24.5 ±i.9 19.6 ±0.7 17.6 ±i.O 32.i ±2.5 1.8 0.8 ±0.1 1.3 ±0.3i.6DMBA-Ad-B8-L22.5 ±2.1 20.1 ±1.3 16.3 ±i.5 30.3 ±1.7 1.9 0.9 ±0.2 1.9 ±0.22.0DMBA-Ad.C5-E25.8 ±2.1 22.6 ±0.4 9.8 ±1.2 20.4 ±2.5 2.i 0.3 ±0.1 0.6 ±0.12.0DMBA-Ad-c@5-L24.7 ±1.2 22.4 ±1.2 17.2 ±2.1 32.2 ±3.4 1.9 0.4 ±O.i 0.7 ±0.3i.8a

Clones transformed by H5tsi 25 were isolated from virally infected 2°RE cultures, derived from i 4-day-gestation Sprague-Dawley RE's as describedpreviously(20)

and in “Materialsand Methods. ‘â€Some cultures were pretreated with carcinogens (BP, 0.05 @zg/ml,or DMBA, 0.05 @og/ml)prior to virus Infection and aresodesignated.E, early passage, <1 0, clones; L, later passage, >25, clones.

b The population-doubling time in the presence or absence of 100 ng of TPA per ml was determined as described in “MaterialsandMethods.―CThe ability of 1 o3 2°RE cells or 1 or 2 x 1 02 transformants to form colonies of >50 cells in the presence or absence of 1 00 ng of TPA per ml was performedasdescribed

in “Materialsand Methods.―d Cells (1 o3, i o@.or 1O@)in low.Ca2@ medium containing 0.4% Noble agar were layered onto 0.8% agar base layers prepared in the same medium. TPAstudieswere

performed the same way except that 100 ng of TPA per ml were incorporated in both the base and overlay layers. Further details can be found in ‘‘MaterialsandMethods.―0

Mean ± SE.

Properties of Adenovirus-transformed RE Cells

Table1

0

EC.,

Ca

a,C.,

Cl)Ca,0C0

a,

a,Cl)

glucose was added. The cells were then iodinated as previouslydescribed (29). The iodinated monolayers were washed twicewith cold PBS containing 2 m@phenylmethylsulfonyl fluoride,and the cells were scraped and pelleted at 1000 x g for 10mm at 4°.The pellet was resuspended in a minimal amount ofSDS-gei sample buffer (30) and sonicated and heated to 100°for I to 2 mm. An equal number of cpm for each cell type waslayered on the gel, and electrophoresis was performed asdescribed by Laemmli (30), using a slab gel apparatus. Theseparating gel had a concentration gradient of acrylamide from15 (bottom) to 7% (top). The tracking dye was permitted tomigrate 9 cm into the separating gel; the gel was removed and

Chart i . Effect of TPA on the SD of normal 2°REcellsand early-passage (<1 0, E) and late-passage (>25, L)H5tsi 25-transformed 2°REcells. Values represent themaximum SD obtained by each cell type. Variations between triplicate determinations were less than 5%.

214E Ad'A18 Ad'E7 BP'Ad'D2 BP'Ad'El 1 DMBA'Ad'AS OMBA'Ad'Be DMBA'Ad'C5

fixed for 2 hr at 37°in 25% isopropyl alcohol-i 0% acetic acid(v/v) and stained overnight with Coomassie brilliant blue. Thegel was destained in I 0% acetic acid, photographed, dried,and pressed onto filter paper. X-Ray film was placed on top ofthe pressed gel, and the film was exposed for 2 to 4 weeks.The autograms were scanned with a Gilford Spectrophotometer250 and charted onto graph paper. To determine the percentage of LETS in each sample, the number of squares on thegraph under the LETS peak of each transformed clone wascounted and compared to that obtained with the LETS peak of2°REcells (whichwas normalizedto 100%).

Con A Agglutinability. Agglutinabilityof 2°REcells and

AUGUST 1979 3053

Research. on January 22, 2019. © 1979 American Association for Cancercancerres.aacrjournals.org Downloaded from

P. B. Fisher et al.

adenovirus transformants by Con A was performed as previously described (22). Subconfluent cultures grown in Blakebottles were detached with 0.02% EDTA, washed twice inCa2@-and Mg2'@-freePBS, and resuspended in PBS to yield 3x 106/mI. Approximately i .5 X 106 cells in 0.5 ml of PBSwere then placed in 35- x i 0-mm tissue culture or multiweilplates. Dishes were set up in triplicate, and one of the followingmaterials was added to 0.5 ml of cells in PBS to yield a finalvolume of 1.0 ml: (a) 0.5 ml of Con A, 200 jsg/ml, in PBS; (b)0.5 ml of Con A, 200 zg/mI, containing 0.5 M a-methyl glucoside in PBS; (c) 0.5 ml of 0.5 M a-methyl glucoside, in PBS; or(d) 0.5 ml of PBS. Agglutination was observed using an inverted-phase contrast microscope after i , 5, 10, 20, 25, and30 mm. The extent of agglutination was described by a 0 to4+ designation based on the average size of clumped cells: 0,clumps of <5 cells; i +, 5 to 10 cells; 2+, 10 to 20 cells; 3+,20 to 30 cells; and 4+, >30 cells.

RESULTS

Growth Properties of 2°RECells and H5tsl 25-transformedRE Cells. Table 1 compares the growth propertiesof severalearly- and late-passage clones of H5tsi 25-transformed REcells to those of normal 2°REcells. All of the late-passage(>25) transformants exhibited reduced population-doublingtimes when compared to normal 2°REcells. There were nosignificant differences in the population-doubling times between late-passage transformants obtained with the virusalone, the virus plus BP, or the virus plus DMBA. On the otherhand, early-passage (<1 0) transformants obtained with BPplus virus or DMBA plus virus had shorter doubling times thandid early-passage transformants obtained with virus alone (Table 1). With prolonged passage, the population-doubling timesof the latter transformants decreased. Addition of i 00 ng ofTPA per ml to normal 2°RE cells or to any of the adenovirustransformants, at either early or late passage, did not significantly alter their population-doubling times.

All of the adenovirus transformants had increased SD's whencompared with 2°REcells (Chart i). The average SD for thetransformed clones was 8.0 ± i .8 cells x i 0°/sq cm asopposed to i .7 cells x i 05/sq cm for 2°REcells. No markeddifferences were apparent between clones transformed byvirus alone or virus plus BP or DMBA. Prolonged passage ofthe same clone led to an increase in SD, except in the case ofBP-AD-D2 (Chart 1 ). Clone BP-Ad-Ei i -L had the highest SD

and also the shortest population-doubling time (Table i ; Charti ). TPA increased the SD of normal 2°REcells by 0.4 x 10°cells/sq cm; it increased the SD of all of the H5tsi 25-transformed clones, at both early and late passage, by approximately i .8 ±0.6 x 10°cells/sq cm (the average increase forall 14 transformants).

Secondary RE cells grew equally well in medium containingio or7.5%FBS,buttheyunderwentonlyoneor2doublingsin medium containing 1% FBS. In contrast, all of the H5tsi 25-transformed clones, except Ad-E7-E, grew continuously in i %FBS. Ad-Al 8-L, Ad-E7-L, and both early and late passages ofthe clones BP-Ad-D2, BP-Ad-Ei 1, DMBA-Ad-A6, and DMBAAd-B8 grew at approximately the same rate in 7.5 or i % FBS.Ad-Ai 8-E, DMBA-Ad-C5-E, and Ad-Al 8-L, and DMBA-Ad-C5-L had higher growth rates in 7.5% FBS than in 1% FBS.Addition of 100 ng of TPA per ml to the medium permitted

2°RE cells and clone AD-E7-E to undergo 4 or 5 population

doublings in 1% FBS.Liquid Cloning Efficiency and Anchorage-independent

Growth of HStsl 25-transformed RE Cells. When grown inmedium containing 7.5% FBS, the transformed clones exhibited a 1.5- to i 3.6-fold greater cloning efficiency in liquidmedium than did normal 2°REcells (Table 1). Although AdAl 8 and Ad-E7 had higher cloning efficiencies at late ratherthan at early passages, no consistent difference in cloningefficiencies of the 3 types of transformants was observed. TPAdid not significantly enhance the cloning efficiency of 2°REcells. it did, however, enhance the cloning efficiencies of all ofthe transformants; on the average, there was a i .7- ±0.3-foldincrease (Table 1). in these studies, the transformants weregrown in low-Ca2@'medium because it is characteristic ofadenovirus-transformed cells to grow best under such conditions (5). Additional studies (not shown here) indicated that innormal Ca2@'medium (i .25 mM)most of the transformants alsohad higher liquid cloning efficiencies than normal 2°REcellsand that their cloning efficiencies were further enhanced byTPA.

Anchorage-independent growth was assayed by plating thetransformed clones in Noble agar containing low-Ca2―mediumwith 7.5% FBS (Table 1; Chart 2). Similar results were obtainedusing 1.25 mM Ca2@medium (data not shown). When studiedin the absence of TPA, 2°REcultures (either in low or 1.25 mMCa2@medium) failed to grow in agar. However, all of the latetransformants grew in agar, with efficiencies varying from 0.1to 6.7%. With most of the transformed clones, the late-passagecells grew better in agar than did the early-passage cells. Infact, when studied at early passage the 2 transformed clones,obtained without exposure to BP or DMBA (Ad-Al 8 and AdE7), failed to form colonies (even at 2 x i 0@ cells/plate) inagar, although after serial passage (late) they both showedappreciable colony formation in agar (Table i ; Chart 2). Thus,with prolonged serial passage there is a spontaneous increasein agar cloning efficiency of the transformants. Variations in the

0Ca,

U

Lu0)CC0C.)a,0)

Chart 2. Effect of TPA on agar cloning efficiency of normal 2°REcells andearly-passage (<1 0, E) and late-passage (>25, L) H5tsl 25-transformed 2°REcells. For details, see Table 1.

3054 CANCER RESEARCH VOL. 39

Research. on January 22, 2019. © 1979 American Association for Cancercancerres.aacrjournals.org Downloaded from

Comparative growth of 2°REcells and H5tsi25-transin agar and agaroseformed

REcells.

Cell typesAgar

cloning effi- Agarose cloning efciency (%)b ficiency (%)Agarose/agar

(cloning efficiency)2°RE

Ad-Ai8-EAd-Ai8-LAd-E7-EAd-E7-LBP-Ad-D2-EBP-Ad-Eii-EDMBA-Ad-A6-EDMBA-Ad-B8-EDMBA-Ad-C5-E<O.OOi

<0.001<0.001 0.2 ±0.0

0.i ±0.0 1.4 ±O.i<0.001 0.2 ±0.0

1.2 ±O.i i.7 ±0.i0.2 ±0.0 6.3 ±0.71.1 ±0.3 2.7 ±O.i3.9 ±0.8 10.2 ±0.60.8 ±O.i 4.3 ±0.50.3 ±0.1 1.0 ±0.10

>20014

>200i.4

3i.52.52.65.43.3

Plasminogen acti2°Rvator

production, LETS, and Con A agglutinability ofE andH5ts125-transformedREcellsCell

typeaPA(% of Con A agglutina

flbrinolysis)b LETS (% of RE)cbilityd2°RE0.6

iOOi+Ad-Ai8-E17.5753+Ad-A18-L18.2543+Ad-E7-Eii.7iO2+Ad-E7-Li4.3i23+BP-Ad-D2-E5.1672+BP-Ad-D2-L1

i .6 57 3+BP-Ad-Eii-E30.5 594+BP-Ad-Eii-L24.2 514+DMBA-Ad-A6-E9.6

603+DMBA-Ad-A6-Li2.i 653+DMBA-Ad-B8-E27.0

362+DMBA-Ad-88-L22.3402+DMBA-Ad-C5-E7.2i 63+DMBA-Ad-C5-L1

2.1 19 3+

Properties of Adenovirus-transformed RE Cells

Table 2size of agar colonies were also apparent. Both early and latepassages of the clones BP-Ad-D2, BP-Ad-Ei 1, and DMBA-AdA6 formed larger colonies than obtained with Ad-Al 8-L, AdE7-L, DMBA-Ad-B8-E, DMBA-Ad-B8-L, DMBA-Ad-C5-E, orDMBA-Ad-C5-L. In addition, with the latter group of clones, the

cloning efficiencies in agar were significantly reduced withinocula of less than 1O@cells/plate.

Addition to the agar of 100 ng of TPA per ml, in both thebase (0.8%) and overlay (0.4%) layers, resulted in a significantincrease in the agar cloning efficiencies of all of the transformants (Table 1; Chart 2). With the i 2 cell types that grew in agarspontaneously, the addition of TPA resulted in a 1.6- to 3.9-fold increase in agar cloning efficiency (2.3 ±0.83). In addition, in the presence of TPA, the colonies were larger andbecame macroscopically visible earlier. The 2 early-passagetransformed clones (Ad-Al 8-E and Ad-E7-E), which did notgrow spontaneously in agar, did form colonies in agar in thepresence of TPA. Despite repeated attempts, however, wewere not able to induce the growth in agar of normal 2°REcellswith TPA.

Separate studies designed to investigate the reversibility ofthe TPA enhancement phenomena indicate that subclones ofBP-Ad-Ei i -E cells isolated directly from agar containing TPAgrow with greater efficiency than did the parental clone whenreseeded in agar in the absence of TPA. Thus, the TPA enhancement appears to be irreversible although it is not yetclear whether this reflects cell selection or other mechanisms.°

Normal 2°REcells failed to form colonies in agarose, evenwhen seeded at 5 x 10°cells/plate in the presence of TPA.All of the transformants, however, grew in agarose in theabsence of TPA (Table 2). Their cloning efficiencies Variedwidely (0.2 to 10.2%), and it appears that at early passage thetransformants obtained with virus plus BP or DMBA grew betterin agarose than did those obtained with virus alone. AlthoughAd-Al 8-E and Ad-E7-E did not grow in agar, they had a lowbut significant growth in agarose. These results indicate thatagarose is less restrictive than agar as a suspension mediumfor monitoring anchorage-independent growth of adenovirustransformed cells. They also suggest that acquisition by thesetransformed cells of the ability to grow in agarose can precedetheir ability to grow in agar. it should be emphasized, however,that absolute cloning efficiencies with the same clone variedwith different batches of Noble agar and Seakem agarose.Therefore, quantitative comparisons were only made on dataobtained within the same experiment. The effects of TPA ongrowth in agarose remain to be studied.

Plasmlnogen Activator Production, LETS, and Con A Agglutination. Plasminogenactivatordata are presentedinTable3. Whereas 2°REcells had negligible levels, all of the transformed clones showed appreciable activity.

The quantity of LETS, associated with the cell surface of thenormal and transformed clones, was assayed by SDS-polyacrylamide gel electrophoresis of iodinated cell surface proteins (Table 3). When compared to the normal 2°REcultures,all of the transformants had a decrease in the amount of LETS.The reduction varied considerably between clones and was inthe range of 25 to 90%. it is of interest that although an earlypassage transformant Ad-E7-E appeared to be ‘‘lesstransformed' â€by some of the criteria described above it had the

5 P. B. Fisher, J. H. Bozzone, and I. B. Weinstein, manuscript in preparation.

a See Table 1 and “Materials and Methods' ‘for designation and description

of H5tsl 25-transformed RE clones.b See Table 1 and ‘‘Materials and Methods' ‘for details on agar cloning assay.C This assay was performed as described in Table 1 and ‘‘Materials and

Methods,‘‘except Seakem agarose was used in the base and overlay layers.

Table3

aSeeTable1and‘‘MaterialsandMethods'‘fordesignationanddescriptionof H5tsl 25-transformed RE clones.

b Intact cell assay for plasminogen activator production was performed asdescribed previously (45) and in ‘‘Materialsand Methods.‘‘Results are averagesfrom triplicate samples assayed after 6 hr. Percentage of fibrin released wasdetermined from the ratio of cpm released in the presence of sample (adjustedfor fibrin released In the presence of serum but in the absence of sample) to thetotal cpm released by trypsin.

C Determination of iodinated LETS by SDS-polyacrylamide gel electrophoresis

was performed as described In ‘‘Materialsand Methods.‘‘Values presented areaverages from 3 separate samples compared with 2°RELETS (which wasnormalized to 100%).

d Con A agglutinability was determined as described previously (22) and in.‘Materialsand Methods. ‘‘i ±, approximately 10% clumps of 5 cells, majority ofclumps <5 cells; 1+ , clumps of 5 to 10 cells; 2+ , clumps of i 0 to 20 cells; 3+,clumps of 20 to 30 cells; and 4 + , clumps of >30 cells.

lowest level of LETS (10%). On the other hand, DMBA-Ad-A6-E and DMBA-Ad-A6-L, which had the highest cloning efficiencies in agar, had appreciable levels of LETS (60 to 65%).

Whereas 2°REcells showed only slight clumping when incubated with Con A, all of the transformed clones showedappreciable agglutination, ranging from 2 to 4+ [clumps of 10to 20, or clumps of >30 cells (Table 3)]. Differences in the timerequired for maximal agglutination for the different clones wereobserved. Ad-Al 8-L and both early and late passages of clonesBP-Ad-D2, BP-Ad-El 1 , and DMBA-Ad-B8 were maximally agglutinated by 10 mm, whereas the other transformed clonesand normal 2°REcells required 15 to 30 mm for maximalagglutination.

No consistent differences between the transformants ob

3055AUGUST1979

Research. on January 22, 2019. © 1979 American Association for Cancercancerres.aacrjournals.org Downloaded from

P. B. Fisher et al.

tamed from cells exposed to virus alone, BP and virus, orDMBA and virus were apparent in terms of levels of PA pro

duction, extent of reduction of LETS, or agglutinability by ConA (Table 3). Nor was there a consistent difference in theseparameters between early- and late-passage clones.

DISCUSSION

The transformation of cells in culture by DNA tumor viruses,such as SV4O, polyoma, and adenovirus, can result in a widespectrum of changes, including altered morphology, alteredgrowth properties (reduced population-doubling time and increased SD), decreased serum requirement, increased production of proteolytic enzymes, altered cell surface composition, increased rate of 2-deoxyglucose transport, loss of actinfilament bundles, and acquisition of anchorage-independentgrowth (9, 25, 36, 37). In the present study, transformantsobtained from 2°REcells exposed to H5tsi 25 alone, or chemical carcinogens plus this virus, exhibited the full range of invitro properties previously associated with viral or chemicaltransformation. We observed, however, a remarkable variationbetween cloned transformants in terms of the degree to whichthey expressed several markers of transformation. Althoughexposure of 2°REcells to BP or DMBA prior to infection withH5tsl 25 increased the number of transformed foci obtained(24), the present study indicates that the phenotypic propertiesof these transformants did not differ in a major or consistentmanner from transformed clones isolated from cells infectedwith virus alone (Tables 1 and 3; Charts 1 and 2). Our data dosuggest that the BP and DMBA treatment may have enhancedthe earlier expression of certain markers, namely, a decreasein population-doubling time and the acquisition of anchorageindependent growth, but this remains to be verified with alarger number of clones.

Although attempts have been made to correlate changes inin vitro properties with in vivo tumorigenicity, exceptions havebeen consistently found (9, 23, 25). In a previous study of REcells transformed by adenovirus type 2 (in the absence ofchemical carcinogen treatment), the expression of individualmarkers of transformation also varied appreciably betweentransformed clones and did not correlate in a simple way withtumorigenicity (25). For example, transformants were obtainedthat had a low cloning efficiency in methyl cellulose and a highlevel of LETS, and yet they were tumorigenic in adult nudemice (9, 25). We have also observed a lack of correlationbetween the degree of expression of individual phenotypicproperties in our transformants. For example, the DMBA-AdA6-L clone had a very high cloning efficiency in agar, a highliquid cloning efficiency, a high SD, and a 35% reduction inLETS when compared to normal 2°RE cells. On the other hand,the Ad-E7-E clone, which did not grow in agar, had an increasein PA synthesis comparable to DMBA-Ad-A6-L and a 90%reduction in LETS. Other examples of dissociation of theexpression of markers of transformation can be found byinspecting the tables and charts. it is of interest that all clonesthat grew in agar also grew in agarose, although the reversewas not true. Attempts to induce tumors in newborn SpragueDawley rats by injecting s.c. 2 x 106 H5tsi 25-transformedcells from early-passage clones described in the present studywere unsuccessful,6 even though some of these clones grew in

6 p B. Fisher and I. B. Weinstein, unpublished data.

agar or agarose with high efficiency.Of particular interest is our finding in the present study that

although TPA does not permit normal 2°REcells to grow inagar it causes an appreciable enhancement of growth in agarof the adenovirus transformants (Table 1; Chart 2). In fact, 2early-passage adenovirus-transformed clones, Ad-Al 8-E andAd-E7-E, were capable of growth in agar only when TPA waspresent. With continued passage, however, these clones spontaneously acquired the ability to grow in agar, but the additionof TPA further enhanced their cloning efficiency. These results,together with other differences in phenotypic properties between early- and late-passage clones, suggest that the morphologically transformed clones that appear within a few weeksfollowing infection of 2°REcells with H5tsl 25 undergo, duringserial in vitro passage, spontaneous progression toward a more, ‘transformed state' â€and that TPA can mimic or accelerate this

process. Previous in vitro studies have indicated that followinga single exposure of cells to chemical carcinogens and serialpassage there is also sequential acquisition of the propertiesof transformation (1, 17). In the case of BP transformation ofSyrian hamster embryo cells, morphological transformation isobserved by 8 days posttreatment and fibrinolytic activity isseen by 14 days posttreatment, and yet growth in agar andtumorigenicity do not occur until 6 or more weeks after exposure to BP (1). The acquisition of anchorage independenceand tumorigenicity following the exposure of guinea pig embryofibrobiasts. (17), rat fetal brain cells (38), rat liver cells (33), ormouse epidermal cells (11, 12) to chemical carcinogens hasalso been found to require extended (in these cases, severalmonths) serial passage in culture. Colburn et al. (1 i ) havefound that a strain of serially passaged epidermal cells, whengrown continually in TPA for 3 weeks, acquired the ability togrow in soft agar, whereas parallel cultures not exposed toTPA did not grow in agar. We are presently investigating themechanism by which TPA enhances the growth of the adenovirus-transformed cells in agar.

It is difficult to explain the phenotypic variations exhibited bydifferent H5tsi 25-transformed clones arising from cultures thatreceived the same initial treatment (virus alone or chemicalcarcinogen plus virus). Possible explanations might include: (a)differences in the number or types of adenovirus genes integrated into the genomes of the different clones; (b) the chromosomal locations of the integrated viral genes; (c) heterogeneity of the cell types present in the normal RE cultures; or (d)instability in the expression of host genes (at the genetic orepigenetic level) in the transformed cells. Evidence has beenpresented indicating that the amount and portion of the adenovirus type 2 genome integrated in rat cell lines does notcorrelate with expression of transformation-associated properties (9, 25) or tumorigenicity in syngeneic rats (25). Althoughother explanations have not been excluded, we favor Hypothesis d since it explains not only the phenomenon of phenotypicdiversity but also why, with serial passage and without furthertreatment, there is a progression in the phenotypic propertiesof the transformants. TPA may potentiate these changes byenhancing the expression of transformation-related phenotypicproperties and/or selectively stimulating the growth of specificvariants arising within a clonal population. Studies are in progress to test this hypothesis.

We are impressed by several similarities between this cellculture transformation system and the carcinogenic process as

3056 CANCERRESEARCHVOL. 39

Research. on January 22, 2019. © 1979 American Association for Cancercancerres.aacrjournals.org Downloaded from

Properties of Adenovirus-transformed RE Cells

and Procedures for Cell, Tissue and Organ Culture, Vol. 3, 625-628.Rockville, Md.: Tissue Culture Association, 1977.

21 . Fisher, P. B., Goldstein, N. I., Bonner, D. P. , Mechlinski, W. , Bryson, V. . andSchaffner, C. P. Toxicity of amphotericin B and its methyl ester towardnormal and tumor cell lines. Cancer Res., 35: i 996-i 999, 1975.

22. Fisher, P. B., Goldstein, N. I., and Bryson, V. Properties of morphologicallyvariant haplold frog cells formed by combined treatment with Mango virusand the polyene antibiotic, mediocidin. In Vitro, 14: 961—965,1978.

23. Fisher, P. B., and Weinstein, I. B. In vitro screening tests for potentialcarcinogens. In: J. M. Sontag (ed), Carcinogens In Industry and Environments. New York: Marcel Dekker, Inc., in press, 1979.

24. Fisher, P. B., Weinstein, I. B., Eisenberg, D., and Ginsberg, H. S. Interactionsbetween adenovirus, a tumor promoter, and chemical carcinogens in transformation of rat embryo cell cultures. Proc. Natl. Acad. Sci. U. S. A., 75:2311—2314,1978.

25. Gallimore, P. H., McDougall, J. K., and Chen, L. B. In vitro traits of adenovirus-transformed cell lines and their relevance to tumorigenicity in nudemice. Cell, 10: 669—678,1977.

26. Ginsberg, H. S., Ensinger, M. J., Kauffman, R. S., Mayer, A. J. , andLundholm, U. Cell transformation: a study of regulation with types 5 and i 2adenovirus temperature-sensitive mutants. Cold Spring Harbor Symp.Quant. Biol., 39: 419-426, 1974.

27. Hecker, E. Cocarcinogens and cocarcinogenesis. In: E. Grundman (ad.),Handbuch der Allgemeinen Pathologie, IV, Vol. 16, pp. 65i—676. Berlin:Springer Verlag, 1975.

28. Hiral, K., Defendi, V., and Diamond, L. Enhancement of simian virus 40transformation and integration by 4-nitrogulnoline-i -oxide. Cancer Res., 34:3497—3500.1974.

29. Hynes, R. 0. Alterations of cell-surface proteins by viral transformation andby proteolysis. Proc. NatI. Acad. Sci. U. S. A., 70: 3i 70-3i 74, 1973.

30. Laemmli, U. K. Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature (Lond.), 22 7: 680—685,1970.

31 . Mayer, A. J., and Ginsberg, H. S. Persistance of type 5 adenovirus DNA incells transformed by a temperature-sensitive mutant, H5tsi 25. Proc. NatI.Acad. Sd. U. S. A., 74: 785-788, 1977.

32. Milo, G. E., Schaller, J. P., and Yohn, D. S. Hormonal modification ofadenovirus transformation of hamster cells in vitro. Cancer Res., 32: 2338-2347, 1972.

33. Montesano, R., Saint-Vincent, L., and Tomatis, L. Malignant transformationin vitro of rat liver cells by dimethyl-nitrosamine and N-methyl-N'-nitrosoguanidine. Br. J. Cancer, 28: 215—220,1973.

34, Pietropaolo, C.. Yamaguchi, N., Weinstein, I. B., and Glick, M. C. Glycopeptides from epithelial cell mutants: temperature sensitive for the transformation phenotype. Int. J. Cancer, 20: 738—747,i977.

35. Pollack, E. J., and Todaro, G. J. Radiation enhancement of SV4Otransformation in 3T3 human cells. Nature (Lend.), 2 19: 519—520,i 968.

36. Pollack, R., Osborn, M., and Weber, K. Patterns of organization of actin andmyosin in normal and transformed cultured cells. Proc. NatI. Acad. Sci. U.S. A., 72: 994-998, 1975.

37, Pollack, R., Risser, R., Conlon, S., and Rifkin, D. Plasminogen activatorproduction accompanies loss of anchorage re9ulation in transformation ofprimary rat embryo cells by simian virus 40. Proc. NatI. Acad. Sd. U. 5. A.,71:4792-4796, 1974.

38. Rajewsky, M. F., Augenlicht, L. H., Blessmann, H., Goth, R., Husler, D. F.,Laerum, 0. D., and Lomakina, L. Y. Nervous-system specific carcinogenesisby ethylnitrosourea in the rat: molecular and cellular aspects. In: H. H. Hiatt,J. D. Watson, and J. A. Winston (eds.), The Origins of Human Cancer, ColdSpring Harbor Conference on Cell Proliferation, Vol. 4, pp. 709—726.ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory, 1977.

39. Stoker, M. Effect of x-Irradiatlon on susceptibility of cells to transformationby polyoma virus. Nature (Lend.), 200: 756—758,1963.

40. Todaro, G. J., and Green, H. Enhancement by thymidine analogs of susceptibillty of cells to transformation by 5V40. Virology, 24: 393-400, 1964.

41 . Van Duuren, B. L. Tumor promoting agents in two-stage carcinogenesis.Prog. Exp. Tumor Res., 11: 31-68, 1969.

42. Weinstein, I. B., Wigler, M., Fisher, P. B., Sisskin, E., and Pietropaolo, C.Cell culture studies on the biologic effects of tumor promoters. In: T. J.Slaga, A. Sivak, and R. K. Boutwell (eds.). Mechanisms of tumor promotionand cocarcinogenesis, Carcinogenesis. vol. 2. pp. 313-333. New York:Raven Press,Inc.,1978.

43, WeinsteIn, I. B., Wigler, M., and Pietropaolo, C. The action of tumor-prometing agents In cell culture. In: H. H. Hiatt, J. D. Watson, and J. A. Winston(eds.). The Origins of Human Cancer, Cold Spring Harbor Conference enCell Proliferation, Vol. 4, pp. 751—772.Cold Spring Harbor, N.Y.. ColdSpring Harbor Laboratory, 1977.

44, Weinstein, I. B., Wigler, M., Yamasaki, H., Lee, L. S., Fisher, P. B., andMufson, A. Regulation of the expression of certain biologic markers ofneopiasia. In: R. W. Ruddon (ed), Biological Markers of Neoplasia: Basicand Applied Aspects, pp. 451 —471. New York: Elsevier-North HollandPublishing Ce., 1978.

45. Wigler, M., and Weinstein, I. B. Tumor promoter induces plasminogenactivator. Nature (Lend.), 259: 232—233,i 976.

3057

it occurs in the intact animal. Both display interactions betweenmultiple types of causative factors (initiating carcinogens, tumor promoters, and viruses), the multistep and progressivenature of the process, and heterogeneity with respect to thetypes of transformants or tumors that emerge. Hopefully, thisand similar cell culture systems will be useful for elucidatingthe mechanisms underlying these fundamental characteristicsof the carcinogenic process.

ACKNOWLEDGMENTS

We thank Douglas Elsenberg and Janet Bozzone for excellent technicalassistance.

REFERENCES

1. Barrett, J. C., Crawford, B. D., Grady, D. L., Hester, L. D., Jones, P. A.,Benedict, W. F., and Ts'o, P. 0. P. Temporal acquisition of enhancedfibrinolytic activity by Syrian hamster embryo cells following treatment withbenzo(a)pyrene. Cancer Res., 37: 3815—3823,1977.

2. Berenblum, I. Origin of the concept of sequential stages of skin carcinogeneels. In: F. F. Becker (ed), Cancer I, pp. 323-344. New York: PlenumPublishing Corp., 197@.

3. Blumberg, P. M., Driedger, P. E., and Rossow, P. W. Effect of a phorbolester on a transformation-sensitive surface protein of chick fibroblasts.Nature (Lend.), 264: 446—447,1976.

4. Boutwell, A. K. The function and mechanism of promoters of carcinogenesis.CRC Crit. Rev. Toxicol., 2: 41 9—443,1974.

5. Casto, B. C. Adenovirus transformation of hamster embryo cells. J. Virol., 4:376-383, 1968.

6. Casto, B. C. Enhancement of adenovirus transformation by treatment ofhamster cells with ultraviolet Irradiation, DNA base analogs anddlbenz(a,h)anthracene. Cancer Res., 33: 402—407,1973.

7, Casto, B. C., Pieczynski, W. J., and DiPaolo, J. A. Enhancement of adenovirus transformation by pretreatment of hamster cells with carcinogenicpolycycllc hydrocarbons. Cancer Res., 33: 819—824,1973.

8. Casto, B. C., Pleczynski, W. J,, Janosko, N., and DiPaolo, J. A. Significanceof treatment interval and DNA repair in the enhancement of viral transfermatlon by chemical carcinogens and mutagens. Chem.-BioI. Interact., 13:105-125, 1976.

9, Chen, L. B.. Gallimore, P. H., and McDougall, J. K. Correlation betweentumor Induction and the large external transformation sensitive protein onthe cell surface. Proc. NatI. Acad. Sd. U. S. A., 73: 3570—3574,1976.

10. Coggin, J. H., Jr. Enhanced virus transformation of hamster embryo cells invitro. J. Vlrol., 3: 458-462, 1969.

11. Colburn, N. H., Vorder Bruegge, W. F., Bates, J. R., Gray, R. H., Rossen, J.D., Kelsey, W. H., and Shimada, T. Correlation of anchorage-Independentgrowth with tumorigenicity of chemically transformed mouse epidermal cells.Cancer Res.,38: 624-634, 1978.

12. Colburn, N. H., Vorder Bruegge, W. F., Bates, J. R., and Yuspa, S. H.Epidermal cell transformation in vitro. In: T. J. Slaga, A. Sivak, and R. K.Boutwell (eds.), Mechanisms of Tumor Promotion of Cocarcinogenesis,Carclnogenesls, Vol. 2, pp. 257—275,New York: Raven Press, 1978.

13. Diamond, L., Knorr, R., and Shimizu, Y. Enhancement of simian virus 40-Induced trasnformatlon of Chinese hamster embryo cells by 4-nitroquinoline1-oxIde. Cancer Res., 34: 2599—2604,1974.

14. Diamond, L., O'Brien, S., Donaldson, C., and Shimizu, Y. Growth stimulationof human diploid fibroblasts by the tumor promoter, 12-0-tetradecanoylphorbol-1 3-acetate. Int. J. Cancer, 13: 721—730,1974.

15. DiPaolo, J. A., Casto, B. C., Miyagi, M., and Popescu, N. C. Viral genomeIntegration In Syrian hamster cells after chemical and virus. Proc. Am.Assoc. Cancer Res., 18: 94, 1977.

i 6. Driedger, P. E., and Blumberg, P. M. The effect of phorbol diesters onchicken embryo fibroblasts. Cancer Res., 37: 3257—3265,1977.

17. Evans, C. H., and DlPaoIo, J. A. Neoplastic transformation of guinea pig fetalcells In culture induced by chemical carcinogens. Cancer Res., 35: 1035—1044, 1975.

18. Fisher, P. B., Bryson, V., and Schaffner, C. P. Polyene macrolide antibioticcytotoxicity and membrane permeability alterations. 2. Phenotypic expression In intraspecific and interspecific somatic cell hybrids. J. Cell. Physiol.,in press, 1979.

19. Fisher, P. B., Flamm, M., Schachter, D., and Weinstein, I. B. Tumor promoters Induce membrane changes detected by fluorescence polarization.Blochem. Biophys. Res. Commun., 86: 1063-i 068, 1979.

20. FIsher, P. B., and Goldstein, N. I. An Improved method for establishingprimary cultures free of contamination. In: V. J. Evans, V. P. Perry, and M.M. vincent (eds.), Tissue Culture Association Manual Techniques, Methods

AUGUST1979

Research. on January 22, 2019. © 1979 American Association for Cancercancerres.aacrjournals.org Downloaded from

1979;39:3051-3057. Cancer Res   Paul B. Fisher, Neil I. Goldstein and I. Bernard Weinstein  in Rat Embryo Cells Transformed by AdenovirusPhenotypic Properties and Tumor Promoter-induced Alterations

  Updated version

  http://cancerres.aacrjournals.org/content/39/8/3051

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/39/8/3051To request permission to re-use all or part of this article, use this link

Research. on January 22, 2019. © 1979 American Association for Cancercancerres.aacrjournals.org Downloaded from