(CANCER RESEARCH 50. 4515-4521. August t. 1990]
Thermal Response of Oncogene-transfected
Rat Cells1
Gloria C. Li,2 C. Clifton Ling, Brian Endlich, and Johnson Y. Mak
Radiation Oncology Research Laboratory, Department of Radiation Oncology, University of California, San Francisco, California 94143
ABSTRACT
Rat embryo cells or Rat-1 fibroblasts were transfected with either an
activated c-myc or a c-Ha-rai from the T24/EJ bladder carcinoma, or
they were cotransfected with both. A gene conferring neomycin or hygromycin resistance was also cotransfected so that independent cell lines
could be selected by growth in medium containing the antibiotic. Certain
isolates from cells transfected with only one type of oncogene were further
transformed by exposure to 600 cGy of 250-kVp X-rays. Successful
transfection and transformation were characterized by altered morphol
ogy, increased plating efficiency, shorter doubling time, longer life span,
foci formation, anchorage-independent
growth, and Southern and North
ern hybridization analysis.
The thermal response of these cells at different stages of oncogenic
transformation was examined by exposing exponentially growing cells to
45°C for 0 to 45 min and measuring cellular survivals using colony
formation assay. We found that cells transfected with myc oncogene,
singly or in combination with ras, were more sensitive to thermal stress.
Aside from that, the cells' thermal sensitivity was not affected by the
degree or the nature of transformation.
INTRODUCTION
In the last decade various kinds of studies have provided
evidence in support of a genetic basis for cancer. Considerable
data indicate the existence of endogenous cellular genes, the
protooncogenes, which have latent oncogenic potential (1, 2).
Normal expression of protooncogenes plays an important role
in cellular functions. However, certain genetic events can acti
vate the protooncogenes, resulting in altered gene products,
changes in the level of gene expression, or both (3, 4). The
abnormal and unregulated expression of these genes may be
causally related to the formation of malignancy (1-4). Many
oncogenes have been identified in human cancer, e.g., Ha-ras
in bladder carcinoma, Ki-ras in ovarian adenocarcinomas, Nmyc in small cell lung cancer, c-erb-B in epidermoid lesions,
and c-abl in chronic myelogenous leukemia (5-10).
The roles of protooncogenes and their aberrant counterparts
in cellular function, growth, and differentiation have been ex
tensively studied. The many known oncogenes can be broadly
grouped into several classes based on the functions and the
locations of their gene products. That the many oncogenes can
be thus classified suggests that the encoded proteins function
via a limited number of mechanisms. For example, oncogenes
can be distinguished on the basis of the nuclear or cytoplasmic
localization of their gene products. The nuclear oncogenes, e.g.,
c-myc, are generally strong in their ability to immortalize cells,
but weak in their ability to promote anchorage independence
of fibroblasts (4, 11). In contrast, the cytoplasmic oncogenes,
e.g., ras oncogene, are inefficient in immortalizing cells, but
Received 9/14/89; revised 1/8/90.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work is supported in part by grants CA31397 (G. C. L., J. Y. M.) and
CA42044 (C. C. L.. B. E.) from the National Cancer Institute, NIH. Department
of Health and Human Services, and by Grant DE-FG03-88ER60695 (C. C. L.,
and B. E.) from the Department of Energy.
2To whom requests for reprints should be addressed, at University of Califor
nia, San Francisco, Department of Radiation Oncology, Radiation Oncology
Research Laboratory, Mission Center 200, Box 0806. San Francisco, CA 941430806.
efficient in inducing anchorage independence (4,12). The mech
anisms of activation of the various oncogenes can also be
different; e.g., c-myc is activated by enhanced expression,
whereas the ras family is activated by gene mutation and the
associated production of mutant proteins. Based on these and
other experimental evidence, it is generally accepted that the
constitutive expression of protooncogenes produces proteins
that mediate normal cell function, and when the regulation of
such expression is disrupted, cellular changes and neoplasia
may result.
Tumorigenesis is believed to be a multistep process. Studies
using animal models suggest two of these steps to be (a)
initiation and (b) promotion (13). Initiation is induced by a
single subthreshold dose of a carcinogen, and promotion is
caused by repeated or multiple treatments with an agent (e.g.,
tumor promoter) which by itself is either noncarcinogenic or
weakly carcinogenic (13). At the cellular level, development of
the transformed phenotype also appears to involve multiple
steps (Footnote 3; Refs. 14 and 15). For example, Kennedy and
coworkers showed that transformation of in vitro cell culture
by chemical carcinogens or X-rays required at least two steps.
The initial step is frequent, caused by mutagenic carcinogens,
and bequeaths to cells a fairly stable heritable characteristic.
The second event, however, is rare and random and has been
conjectured to be akin to a spontaneous mutation, which has a
constant but small probability of occurring each time a cell
divides. The study on the clone size distribution of X-raytransformed C3H10T'/2 cells supports this working hypothesis
(13). At the molecular and genetic level, Land et al. (11) and
others demonstrated that either the myc or the ras oncogene,
by itself, alters the morphology of diploid primary REC,4 but
is unable to induce transformation In vitro (11) or tumors in
animals.5 On the other hand, when myc and ras were cotrans
fected, they cooperated to transform REC which induced tu
mors in nude mice.5
Recently, using ras and myc oncogenes in the cotransfection
of REC, we have generated a large number of cell lines that
exhibited various degrees of transformed phenotype, as assayed
by morphological changes, anchorage-independent growth, and
tumor induction in animals. This system provides a model to
investigate how oncogenes may affect cellular response to chem
ical or physical stresses, as has been demonstrated in a study of
the radiation response of cells at various stages of oncogenic
transformation (16).
Experimental studies with cultured cell lines, transplantable
tumors in small rodents, spontaneous tumors in pet animals,
and clinical trials in humans have shown that hyperthermia, in
combination with radiation or chemotherapeutic agents, offers
promise as a modality for cancer therapy (17). Extensive in
vitro studies indicate that the intra- and extracellular milieu,
such as pH, oxygen tension, and nutrient environment, affects
the thermal sensitivity of cells (17). In addition, the thermal
3 B. Endlich, and C. C. Ling. Three oncogenic events are required to transform
rat embryo cells, manuscript in preparation.
4 The abbreviations used are: REC, rat embryo cells; SDS, sodium dodecyl
sulfate; SSC, standard saline citrate (0.15 M NaCI-0.015 M sodium citrate; Ix
SSC): hsp70, M, 70,000 heat shock protein; hsc70, constitutive form of hsp70.
5 B. Endlich, and C. C. Ling. Tumorigenesis of rat embryo cells depends on
the sequence of raj and myc transfection, manuscript in preparation.
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ONCOGENE TRANSFORMATION AND THERMAL SENSITIVITY
response of cells is affected by a phenomenon termed thermotolérance(18, 19). Specifically, after a nonlethal preheat treat
ment, cells can acquire a transient resistance to a subsequent
thermal stress. This phenomenon has been positively correlated
with the enhanced synthesis of heat shock proteins, belonging
to the M, 70,000 and 27,000 families (20-22). Several studies
have shown that malignant cells are more thermosensitive than
normal cells, although this is not a universal finding (23-29).
Thus, both environmental factors and intrinsic properties may
be important in mediating the cellular response to heat shock.
Understanding factor or factors that affect intrinsic thermal
sensitivities of cells would be of importance.
Certain proteins, the products of specific genes, may influence
the response of cells to stresses. For example, the heat shock
proteins may play a role in thermotolerance (20-22) and the
glycoprotein in drug resistance (30, 31). Thus, it would be of
interest to study the possible influence of oncogene protein
products on thermal response. Aside from the work by
Raaphorst et al. (27), which examined the thermal sensitivity
of ras-transfected mouse embryo cells, there has been no other
report on this subject.
We transfected REC or Rat-1 fibroblasts with c-myc and cHa-ras oncogene, singly or in combination. Certain isolates
from singly transfected cells were further transformed by ex
posure to X-rays. We then determined the thermal response of
these cells at the different stages of oncogenic transformation.
We found that cells transfected with myc oncogene, singly or
in combination with ras, are more sensitive to thermal stress.
MATERIALS AND METHODS
Culture and DNA Transfection of Rodent Cells. The cell culture
condition and transfection procedures have been described previously
(11, 16, 32). Rat embryos from 13- to 15-day-pregnant Fischer rats
were minced, dispersed, trypsinized, and cultured in Dulbecco's modi
fied Eagle's Medium H21 supplemented with 10% fetal bovine serum
and appropriate antibiotics. Freshly prepared REC and subsequent
early passages were used for our transfection experiments.
The RECs were transfected with the human c-Ha-ras oncogene from
the EJ bladder carcinoma and contained in the plasmid pEJIOS, and
the human c-myc oncogene was contained in the retroviral vector
pMV6/c-myc (9, 33). Each vector also contained the neomycin phosphotransferase gene conferring resistance to the G418 antibiotics.
Transfection of the plasmid pEJIOS was performed using calcium
phosphate precipitation, according to the method of Graham and van
der Eb (33). Retroviral infection with pMV6/c-myc was carried out
using polybrene to permeabilize the cells as described by Cepko et al.
(34). Cotransfection of both oncogenes was accomplished with CaPO4
precipitation, with the c-myc contained in the plasmid pM2l (33). After
the transfection procedure, neomycin-resistant cells were selected in
G418 (450 itg/ml)-containing medium for 2 to 3 wk. Drug-resistant
colonies of oncogene-transfected cells were isolated using cloning cyl
inders, trypsinized, and grown to monolayers for further studies. The
cultures were also frozen and stored at -75°C in medium containing
Similar procedures were used for the transfection of Rat-1 fibroblasts
with oncogenes, using a hygromycin resistance gene as a selection
marker. Exponentially growing Rat-1 cells were transfected with human
c-Ha-ras oncogenes or c-myc oncogenes contained in the appropriate
plasmid pEJIOS or pM21, respectively. Transfection of single, or both
oncogenes, was accomplished with CaPO4 precipitation. After the
transfection procedure, drug-resistant cells were selected in medium
containing hygromycin B (500 Mg/ml) for 2 to 3 wk. Selected colonies
of m>'c-transfected cells, or cells cotransfected with myc and ras onco
genes, were isolated using cloning cylinders as described above for the
RECs. Four clones have been isolated from /n>'c-transfected Rat-1 cells
(designated as Rat-l:myc 1, Rat-l:myc 2, Rat-l:myc 3, and Rat-l:myc
4); and six clones are from cells cotransfected with both the ras and the
myc oncogenes (designated as Rat-l:myc+ras 1, Rat-l:myc+ras 2, Ratl:myc+ras 3, etc).
RNA analysis and thermal survival data using Rat-l:myc 1 (ml),
Rat-l:myc 2 (m2), Rat-l:myc 3 (m3), Rat-l:myc+ras 1 (mrl), Ratl:myc+ras 3 (mr3), and Rat-l:myc+ras 6 (mr6) lines are presented in
this study. Results from other transfected Rat-1 cells yield similar
conclusions, but are not included here for the clarity of presentation.
Rat-1 cells transfected with only the hygromycin resistance gene and
selected for hygromycin resistance were used as controls.
Heating. Heating of monolayers of cells was carried out in hot water
baths in specially designed incubators (21, 22). The pH of the medium
overlying the cells was maintained at 7.2 to 7.4 by a regulated gas flow
of air and CO2 and monitored both immediately before, during, and
after heating.
Experiments were performed on Day 3 when the cultures were in
exponential growth. After each treatment, cells were trypsinized,
counted on a Coulter counter, appropriately diluted, and plated. After
10 to 14 days of incubation at 37°C,colonies were fixed, stained, and
counted. Colonies containing more than 50 cells were scored as survi
vors.
Surviving fractions were normalized by the plating efficiency. There
were differences in the plating efficiency among the different cell lines.
Cells containing the myc gene had a plating efficiency between 50 and
70%; raj-containing cells had a plating efficiency of 20 to 40%; and the
primary REC had a plating efficiency of 1 to 5%. The presence of
feeder cells increased the plating efficiency of all the cell lines, with the
effect being most significant for the primary REC cultures. With the
use of feeders, the plating efficiency of the primary REC increased to
10 to 20%. Some experiments were performed with feeders, and results
of surviving fractions plotted as a function of heating time were similar
to those obtained without feeder cells. The plating efficiencies for Rat1 cells, Rat-1 cells transfected with hygromycin resistance gene, or Rat1 cells transfected with myc and/or raÃ-oncogenes were 35 to 80%.
Preparation of DNA and RNA. DNA and RNA were prepared from
the same cells according to the method of Laskie/a/. (35). The medium
was removed from a 150-mm plate of cells (5 x IO6 to 5 x IO7 cells),
and the plate was rinsed with 0.75 ml of cold phosphate-buffered saline.
The cells were scraped into an Eppendorf tube, rinsed with phosphatebuffered saline, centrifuged, and lysed in 0.65% Nonidet P-40-10 mM
Tris (pH 7.5)-150 mM NaCl-1.5 mM MgCl2. The cell lysate was
centrifuged in an Eppendorf centrifuge at maximum speed for 2 min.
The supernatant containing cytoplasmic RNA was removed for further
processing, while the pellet was retained for preparation of DNA. To
prepare RNA, the supernatant was treated with an equal volume of
urea buffer |7 M urea-10 mM Tris (pH 7.5)-10 mM EDTA-350 mM
NaCI-1% SDS], mixed vigorously, extracted twice with phenol, and
then precipitated with ethanol. To prepare DNA, the pellet was resuspended in 1 ml of nuclear lysis buffer [100 mM NaCl-40 mM Tris (pH
7.5)-20 mM EDTA-0.5% SDS]. Proteinase K (10 ¿il
of 10 mg/ml) was
added, and the solution was shaken gently at 50°Cfor 4 to 16 h; phenol
10% dimethyl sulfoxide.
Some isolates of cells transfected with single oncogenes were subse
quently given 600 cGy from a 250-kVp X-ray generator. Colonies
which exhibited morphological changes, as characterized by loss of
contact inhibition and piling up in the center of the colony, were isolated
and grown into confluent cultures for further studies.
All the cell lines used in this study were derived from individual
extracted twice and ethanol precipitated. The DNA pellet was rinsed
clones. Nomenclatures for the various cell lines used in this study are
with 70% ethanol, resuspended in 1 ml of Ix TE[10mM Tris (pH 7.5)as follows: REC:myc, m>'c-transfected REC; REQras A and REC:ras
1 mM EDTA], and digested with 10 ^1 of RNase (10 mg/ml) at 37°C
B, ros-transfected REC clones A and B; REC:myc+ras, REC cotransfor 1 h. The DNA was then extracted twice with phenol and precipitated
fected with myc and ras oncogenes; REC:ras/myc, REC transfected
with ethanol.
first with ras oncogene and subsequently transfected with myc onco
gene; REC:myc/X, X-ray-transformed REC:myc; REC:ras/X, X-raySouthern Hybridization. Hybridization was carried out using a mod
ified version of the Southern technique (36). DNA samples were ditransformed REQras.
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ONCOGENE TRANSFORMATION
AND THERMAL SENSITIVITY
gested with appropriate restriction enzymes, and 10 /jg of each sample
were size fractionated on 1% agarose gels. The DNA was depurinated
and denatured in the gel and transferred to Zeta-Probe membrane
(Biorad) by rapid alkaline transfer. The membranes were air dried or
baked as necessary. Prehybridization was carried out for l h at 37°Cin
0.5 M sodium phosphate-1 mvi EDTA-7% SDS. Probes were labeled
by the hexamer primer method (37), to specific activities of 1 to 3 x
IO8cpm/Vg, and were added to the hybridization reaction and incubated
for 18 h at 60°C.Following hybridization, the filters were washed in
2x SSC-1% SDS, initially at room temperature for 5 min, and then
several more times at 65°Cfor 30 min each. The membranes were air
dried and autoradiographed with Kodak XAR-5 X-ray film and Kodak
"Lightning-plus" intensifier screens at —75°C.
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Northern Hybridization. Gene expression is analyzed by Northern
hybridization. RNA (10 to 20 ^g) denatured with glyoxal (38) was size
fractionated on 1% agarose gels and transferred to Hybond membranes
(Amersham) by capillary transfer in lOx SSC. The membranes were
incubated with approximately IO7cpm of probe in a 10-ml solution of
u
0.25 M sodium phosphate (pH 7.2), 7% SDS, and 10% dextran sulfate
for 16 h at 60°C.Following hybridization, the membranes were washed
several times in 2x SSC-1% SDS at 60°Cfor 30 min and then
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RESULTS
Transfection of a single oncogene into REC results in cells
with partially transformed characteristics. These include alter
ation of morphology, shorter doubling time, longer life span,
and higher plating efficiency. The above alterations are presum
ably due to the transcriptional and translational expression of
the transfected myc or ras oncogene. The typical doubling time
for primary REC is about 48 h; for ras-transfected cells, 25 h;
and for w>'c-transfected cells, 15 h. X-irradiation of cells trans
fected with single oncogene induces further morphological
changes in some of the surviving colonies. Detailed results of
the transfection and transformation studies have been reported
recently (16).
Southern and Northern Hybridization Analysis. The successful
transfection of myc and/or ras oncogenes into REC primarycells and their subsequent expression are verified by Southern
and Northern hybridization analysis. Fig. la shows Southern
hybridization analysis of the transfected cell lines probed with
c-m>rexon 3 fragments. £coRIdigestion of cellular DNA yields
an endogenous myc band of approximately 13 kilobases and a
5.5-kilobase band corresponding to the transfected myc. The
exogenous myc band is clearly present for the cells transfected
with the myc gene, but absent for the primary REC and cells
transfected only with the ras oncogene. The extra bands seen
for clones of REC cotransfected with myc and ras are probably
due to multiple or incomplete integration of the transfected
gene, leading to different restriction fragments with some homology to the myc exon 3 probe. Fig. \b shows Southern
analysis using a c-Ha-ras probe. The BamHl digestion of cel
lular DNA yields endogenous ras bands of 8 and 10 kilobases
and a band of 13 kilobases corresponding to the transfected ras
oncogene. The presence of the ras oncogene in transfected REC
cells is further supported by the antibiotic G418 selection
criteria and the Northern analysis of ras-expression discussed
in the following paragraph.
Expression of the transfected myc and/or ras oncogene is
assessed by probing the cytoplasmic RNA with the respective
oncogene probe. The results are presented in Fig. 2. Fig. 2a
shows the Northern blots of cellular RNA probed with the cmyc oncogene. Successful expression of the c-myc gene yields
mature RNA of 2.3 and 5 kilobases depending on the myc
construct used for the transfection. It is clearly demonstrated
Fig. l. Southern hybridization analysis for rat embryo cells transfected with
myc and/or ras oncogenes. The nomenclatures are as follows: ree. primary rat
embryo cells; m. REC:myc; rA. REC:ras A; rB, REOras B; r/mA, REC:ras/m>c
A; r/niB, REC:ras/myc B; m+rC, REC:myc+ras C; m+rD, REC:myc+ras D; rX,
X-ray-transformed REC:ras; and mX, X-ray-transformed REC:myc. r/mA repre
sents clone A isolated from REC cells transfected first with ras oncogene and
subsequently transfected with myc oncogene; m+rC represents clone C isolated
from REC cells cotransfected with myc and ras oncogenes simultaneously, a,
Southern blot of restriction fragments from EcoR\ digest and probed with c-myc
exon 3. fcoRI digestion yields an endogenous myc band of approximately 13
kilobases and a 5.5-kilobase band corresponding to the transfected myc. h,
restriction fragments from BamHl digest probed with c-Ha-ras. The endogenous
bands are at 8 and 10 kilobases. The fragment from the transfected gene is
expected to be at 13 kilobases. An increased signal is observed for ras-transfected
cell lines but not for the primary REC or REC transfected only with myc oncogene.
that myc expression is minimal for primary REC cells and cells
transfected with only the ras oncogene. As shown in Fig. 2b,
expression of c-Ha-ras yields a band of 1.2 kilobases. This band
is not seen with either the primary REC or with cells transfected
with only the myc oncogene.
Similarly, Northern analysis was carried out for Rat-1 cells
transfected with myc, or with both ras and myc along with a
gene conferring hygromycin resistance. Fig. 3, a and b, shows
expression levels for myc and ras for a number of hygromycinresistant Rat-1 colonies. Fig. 3a shows that only cells trans
fected with myc exhibited myc expression. Likewise, Fig. 3A
shows the same conclusion for ras-transfected cells, that only
cells cotransfected with ras exhibited ras expression. It is also
clearly demonstrated that the expression of c-myc or c-Ha-ras
is absent in the control Rat-1 cells, i.e., those transfected with
hygromycin resistance gene alone. The level of myc expression
varies between individual isolates, and those having various
levels of expression were chosen for further study.
Thermal Response at 45°Cof Primary- and Oncogene-transfected REC. Monolayers of exponentially growing primary REC
or REC transfected with oncogenes were exposed to 45°Cfor
0 to 45 min. Cellular survival probabilities were determined by
colony formation assay.
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ONCOGENE TRANSFORMATION AND THERMAL SENSITIVITY
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Fig. 2. Northern hybridization analysis of cytoplasmic RNA from RECderived cell lines, a, Northern blots probed with c-myc exon 3. Expression of the
transfected myc oncogene yields mature mRNA of 2.3 or 5 kilobases depending
on the myc construct used for the transfcction. b. Northern blots probed with c11.1;<MExpression of the transfected ras gene yields a band at 1.2 kilobases. The
nomenclatures are the same as described in Fig. 1.
b
Fig. 3. Northern hybridization analysis of cytoplasmic RNA from Rat-1 cells
transfected with myc and/or ras oncogenes. The nomenclature is as follows: h,
Rat-1 cells transfected with only the hygromycin resistance gene; ml, Rat-l:myc
1; m2. Rat-1:myc 2; m3. Rat-1:myc 3: mrl, Rat-l:myc+ras 1; mr3, Rat-l:myc+ras
3; and mr6. Rat-l:myc+ras 6. a and ft. Northern blots probed with c-myc exon 3
or c-Ha-ras, respectively.
Fig. 4a presents the surviving fraction as a function of heating
time at 45°Cfor 4 cell lines: two primary REC preparations; a
clonal derivative of REC transfected with myc (REC:myc); and
an X-ray-transformed REC:myc line (REC:myc/X). The two
sets of data for primary REC in Fig. 4 and data from other
experiments (data not shown) indicate that the thermal sensi
tivity of REC from different preparations is relatively constant.
Furthermore, the normalized surviving fractions are independ
ent of the use of feeder cells and the associated changes in
plating efficiency. The incorporation of a myc gene into the
genome of REC appears to alter the cells' thermal sensitivity.
The REC:myc cells are more sensitive to 45°Cheat shock than
are the primary REC cells. A pooled population from REC
cells transfected with c-myc oncogene shows similar increased
thermal sensitivity (data not shown). Moreover, X-ray induced
transformation appears to make REC more thermal resistant.
The survival values after 45°Cheating for 45 min are 2 x 10~2,
2 x 10~\ and 4 x KT4 for REC, REC:myc/X, and REOmyc,
respectively.
Fig. 4b exhibits the surviving fractions as a function of
heating time at 45°Cfor primary REC, REC transfected with
ras (two clones designated as RECrras A and RECrras B), and
X-ray-transformed REC:ras (REC:ras/X). The incorporation
of a ras oncogene into its genome appears to have no discernible
effect on the thermal sensitivity of REC; and similar to that
TIME AT 45'C (min)
15
30
45
TIME AT 45'C (min)
Fig. 4. Surviving fraction at 45*C for primary rat embryo cells (REC) and
REC transfected with the myc and/or ras oncogenes. Exponential growing cells
were exposed to 45°Cfor various times, and survivals were determined by colony
formation assays, a, 45°Csurvivals for two primary rat embryo cell preparations
(REC. •).a clonal derivative of REC transfected with myc (myc, D), and an Xray-transformed REQmyc line (myc/X, O). b, 45'C survivals for two REC
preparations (•):two clones of REC transfected with ras, REOras (ras A and ras
B. O); and an X-ray-transformed REOras (ras/X, O).
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ONCOGENE TRANSFORMATION
AND THERMAL SENSITIVITY
observed for myc-transfected cells, the REC:ras/X cells are
more thermal resistant than the REC:ras cells. For example,
the survival values after a 45°C,45-min treatment are 2 x 10~2,
2 x 1(T2, and 1.5 x 10~' for REC, REOras, and REC:ras/X,
respectively.
The surviving fractions as a function of heating time at 45°C
for primary REC and REC cotransfected with myc and ras
oncogenes are shown in Fig. 5. Four transfected clones were
used for this study: two clones were transformed and tumorigenie (REC:myc+ras); the other two clones were transformed
but not tumorigenic (REC:ras/myc). All these REC are much
more sensitive to thermal stress than are the primary REC,
with a 500- to 1000-fold lower surviving fraction for 45-min
heating at 45°C.They also appear to be more sensitive than
wvc-fansfected REC, with the surviving fraction being 10-fold
lower for 45-min heating at 45°C(compare Figs. 4 and 5).
In Fig. 6 we present the surviving fractions as a function of
heating time at 45°Cfor Rat-1 cells transfected with the myc
or both the myc and the ras oncogenes. As a control, Rat-1
cells were also transfected with only the gene conferring resist
ance to the antibiotic hygromycin B. The thermal sensitivity of
Rat-1 cells was not altered by the incorporation of the hygromycin-resistant gene (data not shown). For clarity, only the
surviving fraction data of the transfected cells are presented in
Fig. 6. In Fig. 6. the cell lines designated ml. m2, and m3 were
transfected with myc; and those designated mrl. mr2, and mr3,
with both myc and ras. Northern analysis indicated expression
of myc in ml, m2, mrl, and mr3, but not in m3 and mr6.
Similar to our results for REC, a correlation between myc
expression and thermal sensitivity is observed. The incorpora
tion of a myc gene into the genome of Rat-1 cells and its
subsequent expression enhances cells' thermal sensitivity.
TIME AT 45°C(min)
Fig. 6. Surviving fractions at 45'C for Rat-1 cells transfected with myc
oncogene MI, A/2, MS and Rat-1 cells cotransfected with myc and ras oncogenes
A//?/, MR3, MR6). Control cells were transfected only with the gene conferring
resistance to the antibiotic hygromycin B H.
sensitivity. Specifically, both REC and Rat-1 cells transfected
with c-myc oncogene become more sensitive to 45°Cheating
than did the parental cells. In contrast, the incorporation of a
ras oncogene into its genome appears to have no significant
effect on the intrinsic thermal sensitivity of REC.
However, when both the ras and myc oncogenes are trans
fected into REC, the thermosensitivity of the cells is further
enhanced over and above that of cells transfected with only the
myc oncogene. X-ray-induced changes of singly transfected
DISCUSSION
REC seem to be accompanied by a decrease in thermal sensitiv
We have shown that the incorporation of certain oncogenes
ity. This observation applied to both ras or myc transfected
into the genome of cells may lead to an alteration in thermal
REC (Fig. 4). In addition, there appears to be no significant
difference in thermal sensitivity whether cells were cotrans
fected with myc and ras simultaneously (tumorigenic), or first
transfected with ras and then followed by myc oncogene (trans
formed but not tumorigenic). Thus, we found no correlation
between thermal sensitivity and the ability of cells to induce
tumors in animals (Fig. 5).
Cell cycle distribution data was obtained for some of these
lines in a previous study (16). While there were some variations
in the cell cycle distribution among the different cell lines, these
were insignificant relative to the effects observed. Specifically,
the proportion of the heat-sensitive S-phase cells was not sig
nificantly higher for cell lines which were thermally sensitive.
Thus, the observed variation in thermal responses was not due
to differences in cell cycle distribution.
The use of the REC system has several advantages for study
ing the effect of oncogene on thermosensitivity. (a) The REC
progenitors are normal diploid cells, (b) Since REC have not
undergone unspecified changes which are associated with estab
lished cell lines, well-defined genetic alteration can be intro
duced, (c) Radiation can be applied to further transform these
cells. Following this approach, cell lines at different stages of
TIME AT 45°C(min)
oncogenic transformation can be produced. These cell lines
Fig. 5. Surviving fractions at 45"C for primary REC and REC cotransfected
provide a model system for systematic studies of the possible
with myc and ras oncogenes. Primary REC (•);two clones, transfected and
effect of oncogenesis on cellular response to various stresses,
tumorigenic. were cotransfected with myc and ras simultaneously (R+M, */. O;
R+M, #2, A); and two clones, transformed but not tumorigenic, were transfected
first with ras and then by the myc oncogenes (A/A/, #/, D; A/A/, #2, V). 45°C such as heat shock, radiation, or chemotherapeutic agents. On
the other hand, primary REC can only be maintained for a few
survivals for REC cells transfected with myc oncogene were shown for compari
son.
passages, sufficient for thermal survival assays, but not long
4519
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ONCOGENE TRANSFORMATION
AND THERMAL SENSITIVITY
enough for transfection, selection, and further survival studies.
Thus, the primary REC system does not permit the use of a
negative control, that is, the transfection by a plasmid contain
ing only the neomycin-resistant gene, but without an activated
oncogene. This absence of a negative control for REC may be
a disadvantage, since we cannot rule out the possibility that the
transfection process itself alters cellular thermosensitivity, or
that a specific subpopulation more efficient in receiving exog
enous DNA is also more thermosensitive. However, the obser
vation that the transfection of the ras gene into REC did not
alter the cells' thermal sensitivity suggests that this negative
control is not necessary. In fact, the ras-transfection experi
ments can be viewed as a negative control, providing a contrast
between the effects of incorporating myc and that of incorpo
rating the ras oncogene.
To complement the study using the REC system, we transfected Rat-1 cell lines with the myc and the ras oncogene, along
with the hydromycin resistance gene. Thermal survival experi
ments yielded similar results as those from REC studies. Spe
cifically, the incorporation of a myc oncogene into the genome
of Rat-1 cells and its subsequent expression enhanced the cells'
protein stimulates the level of appropriately initiated expression
from the human heat shock protein hsp70 promoter. In con
trast, c-myc protein inhibits the level of appropriately initiated
expression from the mouse metallothionein I promoter. Thus,
the c-myc protein can both positively and negatively regulate
gene expression by stimulating or inhibiting transcription from
mammalian promoters in a novel manner (46).
Very little is known as to whether or how c-myc affects hsp70
expression. One conjecture is that the over-production of c-myc
protein stresses cells and activates the hsp70 promoter in a
manner that interferes with the normal heat shock response.
Another speculation involves the competitive synthesis of cmyc protein and the proteins (e.g., hsp70) which affect cellular
response to heat. It has been demonstrated in a viral system
that the enhanced synthesis of viral proteins competes with the
hsp protein synthesis (47). The possible binding of c-myc pro
tein to one or more heat shock proteins to form complexes thus
disrupting the putative protective effects of hsp may be yet
another hypothetical pathway. Recently, Clarke et al. reported
the purification of complexes formed between M, 53,000 pro
tein of the nuclear oncogene p53 and the heat shock proteins
thermal sensitivity. As a negative control, the incorporation of of rat and Escherichia coli cells. These complexes between hsc70
and p53 are dissociated by ATP-mediated reaction, analogous
the hygromycin gene did not modify thermal sensitivity.
Raaphorst et al. (27) have shown that the T24 human H-ras
to the other ATP-driven reaction performed by hsc70, e.g., the
oncogene did not alter the thermal sensitivity of C3H10T'/2
dissociation of clathrin triskelions from coated vesicles (48).
cells. In their study, mouse embryo cells (C3H10T'/2) were While the significance of the p53-hsc70 complex is unknown,
transfected with a plasmid containing the H-ras oncogene and the conservation of p53-heat shock protein interactions and the
specificity of the ATP-dependent dissociation reaction, how
neomycin resistance gene. Five transfected cell clones showing
neomycin resistance were isolated and established as cell lines. ever, suggest that the interaction of heat shock proteins and
Two of these cell lines expressed a normal morphology, while oncogene products may be important in certain cellular func
three showed a transformed morphology. The sensitivity to tions. In any case, the increased thermal sensitivity in cells
hyperthermia of the five transfected cell lines was the same as expressing myc oncogene, but not in cells expressing only ras
that of the normal cell line for temperatures ranging from 42°C oncogene, suggests the importance of the role that nucleus may
to 45°C.Our survival results with ras-transfected REC are play in thermal sensitivity.
consistent with their findings.
Fitzgerald et al. reported alteration in the values of D0 and
the extrapolation number of the X-ray survival curves, when ACKNOWLEDGMENTS
NIH3T3 cells were transformed with human N-ras oncogene
We thank Dr. George M. Hahn and Dr. William Lee for valuable
(39). Similarly, Sklar observed that NIH3T3 cells transfected
discussion, Frederico Gonzales for his expert technical assistance, and
with Ki-ras, H-ras, and N-ras oncogenes have higher extrapo
P. Krechmer for her help in the preparation of this manuscript.
lation numbers and larger D0 of their X-ray survival curve
relative to that of the parental line (40). Most recently. Ling
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Thermal Response of Oncogene-transfected Rat Cells
Gloria C. Li, C. Clifton Ling, Brian Endlich, et al.
Cancer Res 1990;50:4515-4521.
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