[CANCER RESEARCH 41, 629-634, February 1981 ]0008-5472/81 /0041-OOOOS02.00
Parameters Distinguishing Herpes Simplex Virus Type 2-transformedTumorigenic and Nontumorigenic Rat Cells1
Arthur H. Hale,2 Louis S. Kucera, Larry W. Daniel, and Moseley Waite
Departments of Microbiology and Immunology ¡A.H. H., L. S. K.], and Biochemistry ¡L.W. D., M. W.I, Bowman Gray School of Medicine, Wake Forest University,Winston-Salem, North Carolina 27103
ABSTRACT
A newly developed experimental model system was used todetermine in vitro transformation-specific parameters which
correlate with tumorigenicity. The data suggested that clonalherpes simplex virus type 2-transformed syngeneic rat embryocells with intermediate, transformed rat embryo fibroblasts (t-REF-G-1) or high, rat fibrosarcoma tumorigenic potential insyngeneic rats could be differentiated from clonal transformednontumorigenic (t-REF-G-2) and nontransformed rat embryofibroblast cells by their growth to increased saturation densityand cloning efficiency in soft-agar medium. All clonal herpessimplex virus type 2-transformed cells, regardless of tumori-
genie potential, possessed an increased rate of hexose transport and plasminogen activator activity and were less fibro-
blastoid in morphology compared to nontransformed rat embryo fibroblast cells. There was no significant difference in cell-
doubling time or total phospholipid composition between clonaltransformed tumorigenic, nontumorigenic, and nontransformedcells. However, all clonal herpes simplex virus type 2-transformed cell lines possessed a decreased percentage of arach-
idonic acid and an increased percentage of monounsaturatescompared to percentages in nontransformed rat embryo fibroblast cells. Saturation density, cloning efficiency, and tumorigenicity in newborn and adult rats of one nontumorigenic clonalcell line (t-REF-G-2) increased with cell passage in tissueculture. Other transformation-specific biochemical parameters(hexose transport, plasminogen activator activity, and fatty acidcomposition) remained stable with in vitro passage of the clonalcell lines. Five subclonal cell lines established from 12-O-tetradecanoylphorbol-13-acetate-treated clonal t-REF-G-2
(low passage) cells showed increased cloning efficiencies compared to 2 subclonal cell lines from mock-treated cells; also,all 5 subclonal cell lines from 12-O-tetradecanoylphorbol-13-acetate-treated clonal cells were tumorigenic in newborn rats.Subclonal cell lines from mock-treated clonal cells were nontumorigenic. It was clear from the results that the absolutecloning efficiency did not correlate with tumorigenicity. This isan important conclusion, since it suggests that quantitation oftransformation-specific characteristics at a given point in time
will not necessarily be indicative of the tumorigenic potential ofthe cells.
INTRODUCTION
Although the transformation potential of HSV-23 in mamma-,
lian cells was first described by Duff and Rapp (6), no systematic approach has been used to delineate the in vitro parameters of cell transformation which correlate with tumorigenicity.A report by Coppie and McDougall (4) indicated that clones ofHSV-2-transformed hamster cells have distinctive in vitro morphologies and differ from the parent line (333-8-9) and amongthemselves in tumorigenicity, content of HSV-2 genetic infor
mation, and marker chromosomes. The correlation of biochemical and biological cellular alterations with tumorigenicity wasnot examined.
Our laboratory (14) was successful in transforming syngeneic white Buffalo REF with HSV-2. From a single progenitor
culture containing foci of transformed cells, we segregatedclonal transformed lines varying from nontumorigenic to highlytumorigenic. The purpose of this investigation was to comparevarious biochemical and biological parameters of cell transformation (growth kinetics, doubling time, saturation density, cloning efficiency, hexose transport, plasminogen activator activity,and fatty acid and phospholipid composition) with the tumorigenic potential of the clonal cells. Results suggested that clonaltransformed tumorigenic cells differ markedly from transformednontumorigenic cells in cloning efficiency and saturation density. Other transformation-specific parameters did not distinguish transformed tumorigenic from nontumorigenic cells.Transformed nontumorigenic cells progressed to tumorigeniccells during cell passage. This progression was paralleled bya simultaneous increase in saturation density and cloning efficiency. A portion of these data was presented elsewhere (13).
MATERIALS AND METHODS
Cell Cultures and Medium. Secondary REF were obtainedfrom 18-day-old rat embryos (syngeneic white Buffalo strain).REF cells were transformed by photoinactivated HSV-2 (ANGstrain) as described previously (14). HSV-2 clonal lines of
transformed tumorigenic and nontumorigenic cells were derived from a single progenitor culture containing foci of HSV-2-transformed cells by cloning in soft-agar medium (Ref. 14;Chart 1). All cells were passaged in Eagle's minimal essential
medium supplemented with 10% (v/v) fetal calf serum, 10%(v/v) tryptose phosphate broth, 100 units of penicillin per ml,100 jug of streptomycin per ml of medium, 0.22% Na2HCO3and 2 rriM L-glutamine at the time of medium preparation
1Supported in part by USPHS Grants CA 12197, CA 26084, and CA 21146
from the National Cancer Institute and R 807073 from the Environmental Protection Agency.
2 To whom requests for reprints should be addressed.
Received February 11,1980; accepted November 5, 1980.
3 The abbreviations used are: HSV-2, herpes simplex virus type 2; REF, ratembryo fibroblasts; t-REF transformed rat embryo fibroblasts; RFS, rat fibrosarcoma; TRA, ^-O-tetradecanoylphorbol-IS-acetate.
Clonal Transformed Lines t-REF-G-1 t-REF-G-2(Tumorigenic in Newborns) {Nontumorigenic)
Transplantedto
Syngeneic NewbornRats
Tumor Cell Line RFS(Tumorigenic in Newborns
and Adults)
Chart 1. Development and nomenclature of HSV-2-transformed nontumori-
genic and tumorigenic cells.
(hereafter called growth medium). All cell culture reagents werefrom Flow Laboratories, Rockville, Md.
Cell Growth Kinetics, Doubling Time, and Saturation Density. To measure cell growth kinetics, cells were seeded in 35-mm dishes using growth medium and were incubated at 35°.
At various intervals, some of the cultures were harvested bydislodging the cells with 0.25% trypsin:0.5% EDTA and counting the total number of viable cells by trypan blue dye exclusionand light microscopy.
The doubling time was measured during the log phase of cellgrowth by determining the length of time for cells to increasethe total cell number by a factor of 2.
The saturation density was calculated by counting the totalnumber of cells per culture dish after 168 to 216 hr of incubation at 35° and dividing by the total surface area of the
culture dish.Cloning Efficiency. A modification (14) of MacPherson's
agar suspension technique (15) was used to measure thecloning efficiency (18).
Hexose Transport and Plasminogen Activator. The rate ofhexose uptake was measured as described previously (10).The activity of plasminogen activation was measured on 3H-
labeled fibrin plates essentially as described by Weber andFriss (20).
Lipid and Fatty Acid Analysis. Cells, harvested by scraping,were washed in 0.01 M sodium phosphate-buffered saline, pH7.2, by centrifugaron (500 x g for 10 min) and were extractedusing the method of Bligh and Dyer (2) as described by Ames(1). Phospholipids were separated from neutral lipids by chro-
matography on short Unisil columns [approximately 0.3 g ofUnisil (Clarkson Chemical Company, Williamsport, Pa.) in adisposable pipet plugged with glass wool]. The neutral lipidswere eluted with 10 ml of chloroform. The phospholipids wereeluted with 10 ml of methanol. More than 90% of the phospholipids were eluted with methanol. Phospholipids labeled with32P were separated by 2-dimensional thin-layer chromatogra-
phy on Silica Gel G plates. Solvent System 1 consisted ofchloroform:methanol:water (87:31:5, v/v), and Solvent System2 was butanohacetic acidiwater (80:26:26, v/v). The lipidswere visualized by staining with I2 vapor; following evaporationof the \2, the silica gel containing the lipid was scraped into ascintillation vial and was counted using a toluene:Triton X-100:
water (2:1:0.2, v/v) scintillation cocktail. Solvents were purchased from Fisher Chemical Company, Pittsburgh, Pa.
Fatty acids from phospholipids obtained from the methanol
wash of Unisil columns (described above) were transmeth-ylated with methanohsulfuric acid as described by Daniel ef al.(5) and chromatographed as described by Morton ef al. (16).
RESULTS
Cell Growth Kinetics, Doubling Time, and Saturation Density. The development and nomenclature of 2 independenttransformed clonal cell lines and one tumor cell line used inour experiments are outlined in Chart 1. Experiments wereperformed to compare the growth kinetics, doubling time, andsaturation density of nontransformed REF cells with HSV-2-
transformed tumorigenic and nontumorigenic clonal cells during low and high passage. Since nontransformed REF cells aredensity dependent for cell growth," it was necessary to seednontransformed REF cells at a density of 5.0 x 105 cells/35-mm Petri dish compared to 1.0 x 105 cells for the transformed
cells. Results indicated that after a 24-hr lag period, nontransformed and clonal HSV-2-transformed cells have similar doubling times (Chart 2; Table 1). However, saturation densitiesfor transformed tumorigenic cells in newborn animals wereapproximately 5-fold greater than those for transformed nontumorigenic cells (Table 1) and 4-fold greater than those fornontransformed REF cells. The cell-doubling time and saturation density of t-REF-G-1 and RFS clonal transformed lines didnot differ significantly between low and high passages (t-REF-G-1, p < 0.1; RFS, p < 0.2; Table 1). However, transformednontumorigenic t-REF-G-2 cells spontaneously progressed toexpression of tumorigenicity in newborn and adult animalsduring cell passage (Table 1); concomitantly, these clonal cellsincreased in saturation density (p < 0.01).
Since clonal t-REF-G-2 cells increased in saturation density
from low to high passage, we examined the saturation densityof 14 additional subclonal cell lines. Low-passage or high-
passage clonal cells were treated with the tumor promoter TPAor were mock-treated for 48 hr in growth medium to alter the
phenotypic properties of the cells. The cells were washed freeof TPA and monodispersed in soft-agar medium, as described
for measuring cloning efficiency. After 3 weeks of incubation,cell colonies were isolated and established into subclonal celllines. Analysis of the saturation density showed variation butno significant difference between subclonal cell lines from TPA-treated versus mock-treated clonal cells (Table 2). All 5 subclonal cell lines from TPA-treated clonal t-REF-G-2 (low-pas
sage) cells exhibited tumorigenicity, while all the subclonal celllines from TPA-treated and mock-treated high-passage t-REF-G-2 clonal cells were tumorigenic. Subclonal cell lines frommock-treated t-REF-G-2 (low-passage) cells were nontumori
genic (Table 2).The variation in saturation density among the different clonal
or subclonal cell lines suggests that high saturation densityalone is not predictive of tumorigenicity in newborn or adultanimals.
Cloning Efficiency. Experiments were designed to determine whether HSV-2-transformed tumorigenic cells could bedistinguished from transformed nontumorigenic cells by theirability to form colonies in soft-agar medium (anchorage independence). At low passage, t-REF-G-1 and RFS tumorigeniccells formed colonies with efficiencies of 38.9 and 52.7%,
" A. H. Hale, L. S. Kucera, and M. Walte, unpublished data.
respectively, when monodispersed in soft-agar medium (Table
1). At high passage, the cloning efficiency significantly increased to 75.7 and 77.8% for t-REF-G-1 and RFS cells,respectively. In addition to their enhanced ability to form colonies, microscopic examination of cell colonies growing in soft-agar medium suggested that t-REF-G-1 and RFS cells pro
duced colonies with increased numbers of cells per colonycompared to those of t-REF-G-2 cells (data not shown). As
expected, less than 0.002% of nontransformed cells (passages4 to 12) had the capacity to form colonies in soft-agar medium
(Table 1). After passage 12, nontransformed REF cells reach
Chart 2. Growth kinetics of clonal HSV-2-transformed tumorigenic, nontu-morigenic, and nontransformed cells at 35°.Cells were monodispersed in growthmedium, seeded in 35-mm Retri plates, and incubated at 35°.At various times,
some of the cultures were harvested to measure number of viable cells per plate.
Parameters Distinguishing HSV-2-Transformed Cells
a "crisis" and stop dividing. In comparison, the cloning effi
ciency for transformed nontumongenic cells (t-REF-G-2) was
6.7% at low passage. However, during cell passage the cloningefficiency spontaneously progressed to 13.8% (Table 1). Calculation of the cloning efficiency (see legend to Table 1) at 3weeks and again at 5 weeks revealed no significant change inthe efficiency.
Subclonal cell lines from TPA-treated clonal t-REF-G-2 (low-passage and high-passage) cells all showed an increased
cloning efficiency compared to that of subclonal cell lines frommock-treated low-passage or high-passage t-REF-G-2 cells
(Table 2).These results suggest that absolute quantitation of cloning
efficiency alone was not always predictive of tumorigenicity inadult or newborn animals. However, with high cell passage orTPA treatment of clonal t-REF-G-2 cells, a correlation was
observed between cloning efficiency and the tumorigenic potential in newborn or adult animals.
Rate of Uptake of 2-Deoxyglucose and 3- O-Methylglucose.Since change in hexose transport rate has been used tocharacterize virus-transformed cells (7, 11, 19), experimentswere designed to measure the uptake of 2-deoxyglucose and3-O-methylglucose into nontransformed REF and clonal HSV-2-transformed cells. Although the rate of uptake was 10-fold
less in exponentially growing nontransformed REF cells compared to that of exponentially growing HSV-2-transformedcells, there was no difference in rate of uptake of 2-deoxyglucose or 3-O-methylglucose between transformed nontumori-genic (low-passage t-REF-G-2) and transformed tumorigenic(high-passage t-REF-G-2, t-REF-G-1, and RFS) cells (Table 1).
Activity of Plasrninogen Activator. Many lines of transformed tumorigenic cells have been shown to produce theenzyme plasminogen activator (3, 6, 8, 12). Assay for plasmin-ogen activator activity in clonal HSV-2-transformed rat cellsshowed that both low- and high-passage cells converted 3H-
labeled fibrinogen to acid-soluble material at approximately the
same rate (Table 1). The rate of conversion for clonal transformed cells was at least 20-fold higher than the rate for
nontransformed cells. In conclusion, an increased activity of
Table 1Examination of in vitro transformation parameters which can distinguish HSV-2-transformed nontumorigenic from tumorigenic REF
Low passage, 4 to 39; high passage, 40 to 81 ; nontransformed REF cells progressed to senescence after passage 12 and were not tested at high passage.Percentage of 1 x 105 monodispersed cells forming colonies in soft-agar medium.
c Each value represented the average of 6 determinations in nmol/min/mg protein.
Determined by measuring the rate of acid-soluble cpm released per hr per mg protein in the presence of rat serum.8 Includes tumorigenicity data from our published work (14). Newborn. 1 day old: adult, >6 months old. Data are expressed as the number of rats with tumors to
number of rats inoculated with 1 x 106 viable cells s.c.
plasminogen activator was measured for clonal HSV-2-trans-
formed cells, regardless of tumorigenic potential of the cells.Cell Morphologies. A comparison of cell morphologies was
made of nontransformed and clonal HSV-2-transformed ratcells at low- and high-passage levels. At low passage, t-REF-G-2 cells (Fig. 1C) were predominantly fibroblastoid, whereast-REF-G-1 (Fig. 1B) and RFS (Fig. 1D) cells tended to be moreepithelioid. There appeared to be more round retractile cells int-REF-G-1 (Fig. 18) and RFS (Fig. 1D) cultures than in t-REF-G-2 (Fig. 1C) cultures. Nontransformed REF cells (Fig. 1/4)
Table 2Phenotypic properties of subclonal cell lines derived from TPA-treated or mock-
treated HSV-2-transformed cells
Clonal transformed cells were mock-treated or treated with various concentrations (20, 200, or 2000 ng/ml) of TPA in liquid growth medium for 48 hr at 37°.
The cells were washed and monodispersed in soft-agar medium. After 3 weeksof incubation at 37°,cell colonies were isolated. Subclonal cell lines from mock-treated and TPA-treated t-REF-G-2 (high passage) were maintained in liquidgrowth medium for 20 passages without added TPA. while subclonal cell linesfrom TPA-treated t-REF-G-2 (low passage) were either maintained for 20 passages in growth medium with 100 ng TPA/ml (TPA-treated clonal cells) or inmedium without added TPA (mock-treated clonal cells). The cloning efficiencyand tumorigenicity in newborn rats were measured (see "Materials and Methods").
were distinctly fibroblastoid in morphology, and there werevery few round retractile cells. Similar results were obtainedwhen clonal transformed cells were examined for cell morphology at high passage (data not shown). In conclusion,alteration in cell morphology was not a distinguishing propertybetween transformed nontumorigenic and tumorigenic cells.
Phospholipid and Fatty Acid Compositions of Total Lipids.Since alterations in lipid composition are associated with transformed cells (16, 21, 22), 4 different experiments were performed to determine whether any changes in phospholipid andfatty acid compositions were specific for transformation ortumorigenicity. The results of one representative experimentdid not indicate (Table 3) any significant difference in thepercentage of phospholipids in transformed compared to non-transformed cells.
Results of the fatty acid composition of total lipids are presented in Table 4. Regardless of passage level, the weightpercentage of arachidonic acid (20:4) was lower for the clonalHSV-2-transformed cell lines compared to that of the nontrans
formed REF cells. Concomitant with a lower percentage ofarachidonic acid (20:4) was a higher percentage of monoun-
saturates in transformed compared to nontransformed cells.The ratio of oleic acid (18:1) to arachidonic acid (20:4) was atleast 5-fold less for nontransformed REF cells compared tothat of clonal HSV-2-transformed cells. Also, there was a
significant (p < 0.05) decrease in the double bond indexassociated with the clonal transformed cells compared to thatof nontransformed REF cells. The double bond index did notdistinguish low-passage nontumorigenic t-REF-G-2 cells fromhigh-passage tumorigenic t-REF-G-2 cells.
DISCUSSION
The results indicated that, of all the parameters studied, onlycloning efficiency correlated with tumorigenic potential. Thiswas most dramatically demonstrated by the clonal cell line t-REF-G-2 (Table 1) and subclonal cell lines established fromTPA-treated clonal t-REF-G-2 cells (Table 2). At low passage,clonal t-REF-G-2 cells or subclonal cell lines from mock-treated
clonal cells did not form tumors in newborn or adult rats; but,with cell passage or establishment of subclonal cell lines from
Table 3Phospholipid composition of nontransformed REF and HSV-2-transformed rat cells grown to confluence
Cells were grown in the presence of medium supplemented with 32P for 4 days to ensure maximum
labeling of phospholipids. All cultures were at confluence at the time of cell harvest.
TPA-treated clonal cells, the tumorigenic potential and cloningefficiency increased. The clonal cell line t-REF-G-1 exhibited
a slightly different pattern; i.e., at low cell passage, these cellshad a relatively high saturation density and high cloning efficiency, but the cells formed tumors in only 50% of the newbornanimals and formed no tumors in adult animals. At high passage, t-REF-G-1 cells increased in tumorigenic potential andcloning efficiency. In comparison, RFS cells had high saturationdensities which did not change with passage number. RFScloning efficiency was relatively high at both low- and high-
passage number; however, an increase in cloning efficiencydid occur as a function of passage number. These cells formedtumors in both adult and newborn animals, suggesting thatcloning efficiencies greater than 50% correlate with tumori-genicity regardless of passage number. Although the absolutepredictive ability of the transformation-specific parameters is
limited, from our data there may be the suggestion that upperand lower limits may be set. The observation that a cell line hasa low growth potential in soft agar cannot be interpreted toindicate that the cells will not form tumors. However, a lowerlimit might be established; i.e., cells with a low cloning efficiency (2 to 12%) may not be able to form tumors (e.g., t-REF-G-2, low passage; subclonal cell lines from mock-treated clonalt-REF-G-2, low passage). Cell lines with higher cloning efficiencies (>12%) most probably will form tumors (e.g., t-REF-G-1 , high passage; RFS, high and low passage; subclonal celllines from TPA-treated clonal t-REF-G-2, low passage).
Evaluation of various biochemical parameters [rate of hexoseuptake, activity of plasminogen activator, and changes in theratio of arachidonic acid to oleic acid (Table 3)] indicated thatall of the biochemical parameters tested are transformationspecific. However, none of the biochemical parameters correlated in any way with the tumorigenic potential of these celllines.
The transformation-specific changes associated with clonalHSV-2-transformed REF were not detected in nontransformed
REF cells (Table 1). After 12 to 15 passages in tissue culture,REF cells reach a "crisis" and stop dividing. These data agree
with the general concept that REF maintain a stable karyotype,and rat cells are resistant to spontaneous transformation afterpassage in vitro (9). In contrast, cultured mouse or hamstercells are relatively unstable in karyotype and expression ofendogenous virus genetic information, and these rodent celltypes can spontaneously transform after passage in tissueculture (17).
ACKNOWLEDGMENTS
We acknowledge expert technical assistance from Iris Edwards and LynnKing. Appreciation is extended to Dr. Douglas Lyles for reviewing the manuscript.
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Fig. 1. Morphology of (A) nontransformed REF. (B) t-REF-G-1, (C) t-REF-G-2, and (O) RFS cells after fixationwith 95% ethanol and staining with 1% crystal violet in95% ethanol. x 125.
1981;41:629-634. Cancer Res Arthur H. Hale, Louis S. Kucera, Larry W. Daniel, et al. 2-transformed Tumorigenic and Nontumorigenic Rat CellsParameters Distinguishing Herpes Simplex Virus Type
